Dissertations / Theses on the topic 'TCA'

This PhD project explored the role of tricarboxylic acid (TCA) cycle enzymes in regulating Chinese hamster ovary (CHO) cell metabolism with respect to growth and recombinant protein expression. It was hypothesised that regulation of central metabolism can influence CHO cell productivity in terms of biomass and protein production. Thus the aim of these studies was to identify the key metabolic reactions of the TCA cycle associated with growth and protein expression in CHO cells. The gene expression of all TCA cycle genes was independently knocked-down using RNAi technology. The small interfering RNA (siRNA) mediated silencing of 11 TCA cycle genes significantly reduced cellular growth along with a decline in adenylate energy charges and an increase in catabolic reduction charges. The gene profiling of glucose and amino acid metabolism (not targeted by siRNA) suggested siRNA mediated knock-down of targeted TCA cycle genes led to cellular stress along with an enhanced rate of glycolysis leading to channelling of glucose for the generation of pyruvate. For the purpose of estimating intracellular metabolites, quenching and extraction method using ammonium bicarbonate and methanol was optimised to use with UCB CHO-K1 cell line and static transient siRNA transfections. A gas chromatography-mass spectrometry (GC-MS) analysis post-silencing of the aconitase gene, which catalyses the conversion of citrate to isocitrate in the TCA cycle, yielded higher MS peak intensities of at least four metabolites (gluconic acid, lysine, threonine and leucine) 72 h post-transfection in comparison to the controls. Transient knock-down of gene expression of seven TCA cycle genes in a recombinant stable cell line (expressing a rabbit monoclonal antibody) reduced cellular growth and altered the energy charges leading to a decline in antibody expression. Although silencing of the pyruvate dehydrogenase E1 gene, which is the component of the pyruvate dehydrogenase complex connecting glycolysis to the TCA cycle, did not affect cell viability, a reduction in antibody expression was recorded. Seven TCA cycle genes which demonstrated the most significant effect on cellular growth and energy charges were transiently over-expressed along with a monoclonal antibody in CHO-K1 cells with addition of their corresponding preceding intermediates. No differences in protein expression and cell specific productivity were observed compared to the control transfections. These results could be due to limitations of the effects of transient transfections for enhancing the metabolic activity of CHO cells. The aconitase gene demonstrated the most significant effect on CHO cell growth and proliferation in this study, therefore this gene was proposed as a novel selection marker for a metabolic selection system for the generation of recombinant therapeutics. This PhD project also established metabolite analysis tools and siRNA protocols for future metabolomic studies for investigating the intracellular CHO metabolism. The findings validated the hypothesis that TCA cycle plays an important role in CHO cell growth and recombinant protein production. The key metabolic genes affecting cellular growth and altering energy metabolism can be further explored for generation of an energy efficient CHO host-cell line (by over-expression of key TCA cycle genes) for enhanced recombinant protein production.

Submitted by Rosemilda Lima (rofriarq@ig.com.br) on 2018-09-13T11:02:44Z No. of bitstreams: 1 Políticas culturais, democratização e acesso à cultura o Domingo no TCA - Rosemilda Mendes Lima.pdf: 9936430 bytes, checksum: 6c7af37d5f7966eab7962893672eff89 (MD5)<br>Approved for entry into archive by Setor de Periódicos (per_macedocosta@ufba.br) on 2018-09-13T20:42:07Z (GMT) No. of bitstreams: 1 Políticas culturais, democratização e acesso à cultura o Domingo no TCA - Rosemilda Mendes Lima.pdf: 9936430 bytes, checksum: 6c7af37d5f7966eab7962893672eff89 (MD5)<br>Made available in DSpace on 2018-09-13T20:42:07Z (GMT). No. of bitstreams: 1 Políticas culturais, democratização e acesso à cultura o Domingo no TCA - Rosemilda Mendes Lima.pdf: 9936430 bytes, checksum: 6c7af37d5f7966eab7962893672eff89 (MD5)<br>A dissertação Políticas culturais, democratização e acesso a cultura: O Domingo no TCA tem como objeto de estudo o projeto Domingo no TCA, que visa uma maior democratização do acesso ao Teatro Castro Alves (TCA) e a sua programação. Criado em um contexto de mudanças nas políticas públicas federais e estaduais, o projeto é realizado há quase dez anos pelo TCA, maior teatro do estado, vinculado à Secretaria de Cultura (Secult) e à Fundação Cultural do Estado da Bahia (Funceb). O Domingo no TCA acontece na Sala Principal do TCA, que tem capacidade para 1.554 espectadores, com ingressos a preço simbólico de R$ 1,00 (inteira) e vem atraindo mensalmente, nas manhãs de domingo, um público numeroso e eclético, formado por adultos, crianças e idosos, oriundos dos mais diversos bairros de Salvador. De 2007 a 2015, foram realizadas 112 apresentações de 104 espetáculos de várias linguagens artísticas. A pesquisa para esta dissertação foi realizada utilizando-se dos registros do Sistema TCA, relatórios institucionais, pesquisas acadêmicas e entrevistas com os atores envolvidos, desde os principais gestores da Secult, aos artistas, produtores e público em geral. O objetivo foi analisar o projeto enquanto instrumento de políticas públicas de cultura e verificar a sua importância para cena e as políticas culturais baianas. A dissertação foi estruturada em três capítulos, de forma que, no primeiro momento, sejam abordados temas conceituais da área da cultura, passando depois ao estudo do Teatro Castro Alves em si e do projeto Domingo no TCA.<br>The dissertation Cultural policies, democratization and access to culture: Domingo no TCA has as object of study the Domingo no TCA (Sunday at TCA) project, which seeks to provide a greater democratization of access to the Castro Alves Theater (TCA) and its events. Created in a context of changes in federal and state public policies, the project is being carried out for almost ten years by the TCA, Bahia's largest theater, linked to the Secretariat of Culture (Secult) and the Bahia State Cultural Foundation (Funceb). The Domingo no TCA presentations take place at the theater's Main Hall, which can accommodate up to 1,554 spectators, with tickets costing a symbolic price of R$ 1.00 (full price). Monthly, on Sunday mornings, they have been attracting a large and eclectic audience consisting of adults, children and the elderly, from all kinds of neighborhoods of Salvador. From 2007 to 2015, there were 112 presentations of 104 different shows from several art forms. The research for this dissertation was carried out using the records of the TCA System, institutional reports, academic research and interviews with the people involved, from the main managers of Secult, to the artists, producers and the public in general. The objective was to analyze the project as an instrument of public policies of culture and to verify its importance for the cultural scene and policies of Bahia. The dissertation was structured in three chapters, such that in the first moment conceptual themes related to culture were approached, then going to the study of the Castro Alves Theater itself and the Domingo no TCA project.<br>La disertación Políticas culturales, democratización y acceso a la cultura: El Domingo en TCA tiene como objeto de estudio el proyecto Domingo no TCA (Domingo en TCA), que busca una mayor democratización del acceso al Teatro Castro Alves (TCA) y su programación. Creado en un contexto de cambios en las políticas públicas federales y estatales, el proyecto es realizado desde hace casi diez años por el TCA, el teatro más grande de Bahía, vinculado a la Secretaría de Cultura (Secult) y la Fundación Cultural del Estado de Bahía (Funceb). Las presentaciones del Domingo no TCA tienen lugar en la sala principal del teatro, que tiene capacidad para 1554 espectadores, con entradas a un precio simbólico de R$ 1,00 (precio entero), y vienen atrayendo mensualmente, en las mañanas de domingo, un público numeroso y ecléctico, formado por adultos, niños y ancianos, oriundos de los más diversos barrios de Salvador. De 2007 a 2015, se llevaron a cabo 112 presentaciones de 104 espectáculos de diversos lenguajes artísticos. La investigación para esta disertación se llevó a cabo utilizando los registros del Sistema TCA, informes institucionales, investigaciones académicas y entrevistas con las personas implicadas, desde los principales gestores de Secult, hasta los artistas, productores y el público en general. El objetivo fue analizar el proyecto como un instrumento de políticas públicas de cultura y verificar su importancia para la escena y las políticas culturales de Bahía. La disertación se estructuró en tres capítulos, de modo que en un primer momento sean abordados temas conceptuales de la cultura, pasando en seguida al estudio del propio Teatro Castro Alves y del proyecto Domingo no TCA.

Genetic mapping of pDS15 revealed that this cosmid clone carries the Sinorhizobium meliloti TCA cycle genes mdh, sucCDAB, sdhAB and part of lpdA. Three genes (mdh, sucC , and sucA) were completely sequenced and submitted to GenBank. The nucleotide and amino acid sequences of the TCA cycle genes encoded on pDS15 were aligned and found to be highly homologous with other closely related rhizobial species. S. meliloti cells grown in LBmc express the mdh-sucCDAB operon as one transcript, based on RT-PCR results. Alternative sigma factor sigma54 was not found to have a role in mdh-sucCDAB expression. Despite considerable effort, we have not been able to isolate sucA mutants via random transposon Tn5tac1 mutagenesis to date. Homologous recombination between a plasmid-borne sucA::Tn5 and wild-type S. meliloti sucA failed to generate a bona fide mutant, as revealed by Southern blot analysis. Plasmid pDS15 was mutagenized with transposons Tn5, Tn5tac1, and Tn5-B20. Three Tn5-B20 insertions were mapped to mdh, sucD, and sucA respectively, and preliminary gene expression studies were done.

In the symbiotic association between S. meliloti and its plant host, the nutrient exchange from plant to bacteroid is in the form of C4-dicarboxylic acids such as malate. These compounds directly enter the TCA cycle and the derived energy is used for the nitrogen-fixation process. Malate dehydrogenase (mdh), the two subunits of succinyl-CoA synthetase (sucCD), and two of the three subunits of 2-oxoglutarate dehydrogenase (sucAB) are encoded as an operon with the order mdh-sucCDAB. The expression of this operon is controlled by a single promoter found directly upstream of mdh. The transcrptional start site was mapped (a guanine residue at postion -63) and RT-PCR demonstrated that expression is as one polycistronic message in cells grown in LBmc. Transcriptional lacZ-gene fusions to mdh, sucD and sucA demonstrated that the mdh promoter is under catabolite control as evidenced by the change in ß-galactosidase expression depending on the carbon-source. Expression was highest with acetate followed closely by arabinose and glutamate but lowest with pyruvate as sole carbon-source. MDH was purified, N-terminal sequenced and kinetic assays were performed to determine Km, Vmax. A pH 10 was found to be the optimal for MDH. 2-oxoglutarate exhibited competitive inhibition on MDH. The annotated genome of S. meliloti contains two alleles (sucB), one chromosomal and one on megaplasmid pSymB, which putatively encode the E2 subunit of 2-oxoglutarate dehydrogenase. Only the chromosomally-borne allele was found to be a functional sucB. Lastly, results of a study of the E3 subunits of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chained keto-acid dehydrogenase were presented.<br>Dans la relation symbiotique entre S. meliloti et la plante M. sativa, l'apport en nutriments de la plante vers les bacteroids se fait sous la forme d'acides C4-dicarboxiliques tel que le malate. Ces composés entrent directement dans le cycle de Krebs et l'énergie produite est utilisée pour la fixation d'azote. Malate déhydrogénase (mdh), les deux composantes de succinyl-CoA synthèse (sucCD), et deux des trois composantes de 2-oxoglutarate déhydrogénase (sucAB), sont inscrites en un opéron dans l'ordre qui suit ; mdh-sucCDAB. L'expression de cet opéron est contrôlée par un seul promoteur situé directement en amont de mdh. Le site de début de transcription a été identifié et RT-PCR a démontré la nature polycistronique de l'expression dans les cellules provenant de bactéries cultivées avec LBmc. Fusions transcriptionelles aux gene-lacZ de mdh, sucD et sucA ont été utilisées pour déterminer les niveaux relatifs d'expression dans des cultures provenant de médiums minimaux contenant des sources de carbone spécifiques. MDH a été purifié, le N-terminal séquencé, et des dosages cinétiques ont été performés pour déterminer Km, Vmax, le pH optimal, et l'effet des inhibiteurs allostériques. Le génome annoté de S. meliloti contient deux allèles qui encodent supposément les composantes E2 (sucB) de 2-oxoglutarate déhydrogénase. Un seul des allèles a démontré être une version fonctionnelle de sucB. Finalement, les résultats d'une étude sur les composantes E3 de pyruvate déhydrogénase, sur 2-oxoglutarate déhydrogénase et sur déhydrogénase de kéto-acide de chaines ramifiées seront présentés.

O principal objectivo deste trabalho consistiu na avaliação de processos de redução/eliminação de TCA em amostras de cortiça, nomeadamente rolhas e discos. Os processos de redução/eliminação de TCA em amostras de cortiça foram avaliados com recurso a sistemas de extracção simples e exaustiva sólido-líquido (Soxhlet), em diferentes condições experimentais (sistema extractor de H20/EtOH (88:12 e 80:20)), com e sem substituição de solvente, presença e ausência de adsorvente. O aumento da percentagem de etanol no sistema extractor revelou-se de extrema importância, pois além de permitir a extracção eficaz de TCA das amostras, pressupõe que após o tratamento as rolhas e os discos não libertem TCA quando colocados em contacto com soluções de menores concentrações alcoólicas (e.g. vinhos, espumantes). O carvão activado revelou uma enorme capacidade adsorvente para o TCA o que potencia futuras aplicações deste adsorvente associado a sistemas extractivos; ABSTRACT: The main objective of this study was to evaluate processes of reduction/elimination of TCA in cork samples, including stoppers and discs. The processes of reduction I elimination of TCA in cork samples were evaluated using a solid-liquid simple and exhaustive extraction (Soxhlet) systems under different experimental conditions (extractant system H20 I EtOH (88:12 to 80:20), with and without replacing the solvent, presence or absence of adsorbents). The increase in the percentage of ethanol in the extractor system proved to be extremely important, because besides allowing efficient extraction of TCA, it assumes that cork stoppers and discs samples after treatment, do not release TCA when placed in contact with solutions of lower alcoholic concentrations (e.g. wine, sparkling wine). The activated carbon adsorbent showed an enormous capacity for TCA trapping which potentiates future applications of this adsorbent in extractive systems.

Genetic characterization of the mdh-sucCDAB operon, encoding enzymes of the TCA cycle, was performed. Isolation of a sucA knock-out mutant was attempted through homologous recombination with plasmids carrying a mutated fragment of the sucA gene. Several strains were screened for the expected phenotype of a 2-OGD mutant. Carbon-source utilization phenotype of the tested strains did not reveal highly dissimilar growth patterns in comparison to wild-type levels. PCR amplification of these strains did not yield the expected band size of a mutant copy of the gene. Furthermore, Southern blot analysis of the putative mutants was inconclusive in confirming their identity. Therefore, a sucA mutant was not successfully isolated. Expression of TCA cycle genes was examined under different growth conditions through qRT-PCR analysis. The sucA gene did not reveal any increase in transcript levels relative to sucD, under all tested conditions. This indicated the possible absence of a promoter independently controlling its expression. Other TCA cycle genes were differentially expressed in mdh and sucB mutants in comparison to wild type. Expression patterns did not appear to vary greatly across the tested carbon sources. Comparison of expression of TCA cycle genes in mutants, relative to wild type, revealed significant induction of sucB with arabinose in its respective knock-out mutant. Examination of transcript levels in bacteroids would elucidate the gene expression patterns observed in symbiotic conditions. In addition, further investigation of the nature of a sucA mutation will be required, to determine the possibility of a lethal phenotype.<br>La caractérisation génétique de l'opéron mdh-sucCDAB qui encode des enzymes du cycle des acides tricarboxylique, a été effectuée. Isolement d'un mutant de sucA a été tentée par recombinaison homologue avec des plasmides portant une insertion dans le gène. Plusieurs souches ont été criblées pour le phénotype attendu d'un mutant de la déshydrogénase de 2-oxoglutarate. Le phénotype d'utilisation de carbone des souches testées n'ont pas révélé de modèles de croissance très dissemblables par rapport au phénotype sauvage. L'amplification par PCR de ces souches n'a pas donné la taille de la bande attendue d'une copie mutant du gène. L'analyse par Southern blot des mutants putatifs n'a pas été concluante pour confirmer leur identité. Par conséquent, l'isolement d'un mutant de sucA a échoué. L'expression des gènes du cycle des acides tricarboxylique a été examinée avec des conditions différentes grâce à qRT-PCR. L'inspection d'expression génétique de sucA n'a pas révélé une augmentation par rapport à la transcription de sucD. Cela indique une possibilité de l'absence d'un promoteur indépendant contrôlant son expression. D'autres gènes du cycle des acides tricarboxyliques sont exprimés différentiellement par les mutants de mdh et de sucB, en comparaison au type sauvage. Les profils d'expression ne semblent pas varier considérablement selon les sources de carbone testées. Comparaison de l'expression de ses gènes a révélé induction significative de sucB avec l'arabinose dans se mutant respectif. L'observation de transcription dans les bactéroïdes serait élucider l'expression dans des conditions symbiotique. Une enquête plus approfondie d'une mutation de sucA sera nécessaire, afin de déterminer la possibilité d'un phénotype résultant mortelle.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Jurídicas. Programa de Pós-Graduação em Direito<br>Made available in DSpace on 2012-10-22T23:20:26Z (GMT). No. of bitstreams: 1 248842.pdf: 1585576 bytes, checksum: c14577e63a4fa73dd71d9e251cd5927d (MD5)<br>A presente dissertação de Mestrado tem como tema central o Tratado de Cooperação Amazônica (TCA) e a sustentabilidade da Região MAP, no período compreendido entre 1992 a 2002. O objetivo é o de verificar se, no período estudado, o Tratado de Cooperação Amazônica, assinado por oito países sul-americanos, dentre eles Peru, Brasil e Bolívia, apresentou-se como um mecanismo efetivo de promoção da sustentabilidade da Região MAP # uma região-fronteira, situada na Amazônia Sul-Ocidental e que compreende o Estado do Acre (BRA) e os Departamentos de Madre de Dios (PER) e Pando (BOL). Para a realização do trabalho, foi feita uma ampla pesquisa monográfica compreendendo, sobretudo, a questão da sustentabilidade amazônica e a evolução institucional do Tratado. A partir da década de 1990, o Tratado de Cooperação Amazônica, adota o desenvolvimento sustentável como tema estratégico, constituindo-se a sua promoção em sua grande missão. Contudo, em razão da sua fragilidade institucional, das políticas nacionais e regionais dos países signatários, contraditórias em relação ao princípio da sustentabilidade, bem como da realidade socioambiental da Região MAP no início do século XXI - que gerou iniciativas locais de cooperação, alternativas ao modelo tradicional de cooperação representado pelo TCA - o discurso de sustentabilidade adotado pelo TCA é um discurso que se apresenta contraditório em relação às práticas das políticas de cooperação amazônica e dos seus países signatários. La presente disertación de Maestría tiene como tema central el Tratado de Cooperación Amazónica (TC 1992 a 2002. El objetivo es verificar se, en el período estudiado, el Tratado de Cooperación Amazónica, asignado por ocho países suramericanos, entre ellos Perú, Brasil y Bolivia, se mostró como un mecanismo efectivo de promoción de la sustentabilidad de la Región MAP # una región-frontera, situada en la Amazonía Sur-Occidental y que comprende el Estado de Acre (BRA) y los Departamentos de Madre de Dios (PER) e Pando (BOL). Para la realización del trabajo, se ha hecha una amplia pesquisa monográfica comprendiendo, sobretodo, a la cuestión de la sustentabilidad amazónica y la evolución institucional del Tratado. A partir de la década de 1990, el Tratado de Cooperación Amazónica adopta el desarrollo sostenible como tema estratégico, constituyendo su promoción en su gran misión. Sin embargo, en razón de su fragilidad institucional, de las políticas nacionales y regionales de los países signatarios, pontradictorias en relación al principio de la sustentabilidad, así como de la realidad socioambiental de la Región MAP en el inicio del siglo XXI - que ha generado iniciativas locales de cooperación, alternativas al modelo tradicional de cooperación representado por el TCA # el discurso de sustentabilidade adoptado por el TCA es un discurso que se muestra contradictorio en relación a las prácticas de las políticas de cooperación amazónica e de sus países signatarios.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-08-18T17:50:06Z No. of bitstreams: 1 texto completo.pdf: 6874332 bytes, checksum: 3e055e6f18562c6805d6dba9dcef9c86 (MD5)<br>Made available in DSpace on 2016-08-18T17:50:06Z (GMT). No. of bitstreams: 1 texto completo.pdf: 6874332 bytes, checksum: 3e055e6f18562c6805d6dba9dcef9c86 (MD5) Previous issue date: 2015-07-17<br>Fundação de Amparo à Pesquisa do Estado de Minas Gerais<br>Mitocondrias vegetais estão envolvidas em vários processos chaves da célula, vão além da produção de energica, tais como morte celular programada, amadurecimento de frutos, ou mesmo aqueles processos dependente de luz como fotossíntese e fotorrespiração. Dessa forma, aquisição mitocondrial pela célula hospedeira trouxe avanços para as atuais células vegetais: desde a manutenção de diversas vias metabólicas que incluem o metabolismo energético bem como processos de bissíntese de lipidios, nucleotidios e vitaminas. No tocante ao metabolismo energ- etico, destaca-se a herança do ciclo do ácido tricarboxílico. Este ciclo é uma via essencial relacionada com a produção de poder redutor (NADH e FADH 2 ), assimilação de nitrogênio e otimização da fotssíntese. Acredita-se que o ciclo do ácido tricarboxílico oprerasse como passos isoladados antes do processo endossimbiótico e somente após a aquisição da mitocôndria resultou que aquele organizar-se e atuasse como uma via cíclica. O cíclo do ácido tricarboxílico é composto por oito enzimas. Contudo, cada enzima é codificada por vários genes os quais são endereçados para diversos compartimetos celulares e, não somente, mitocôndrias. Essas enziimas locallizadas em diferentes comparimentos subcelulares acarretaram em uma possível ampla conecção entre mitocôndrias e outras organelas (peroxissomos e cloroplastos) permitindo fluxos alternativos dos intermediários do ciclo cujo resultado alterou seu funcionamento para um não convencional modo não cíclico. É bastante aceito que sob estresses, no quais reduzem os níveis de carboidratos. O ciclo do ácido tricarboxílico pode funcionar no modo não cíclico, devido a perda de esquelos carbônico que entram se fazendo necessário ser alimentado por reações anapleuróticas. Portanto, aminoácidos tornam-se fundamentais para suprir a respiração e síntese de ATP sob tais situações. Fortes evidencias demonstraram que aminoácidos de cadeia ramificada (BCAA) e lisina podem fornecer elétrons para o sistema a cadeia de transporte de elétrons mitocondrial pela ação do sistema flavoproteína de transferência de elétrons (ETF)- ETF: ubiquinona oxidorredutase (ETF/ETFQO). Em plantas, duas enzimas: Isovaleril-CoA desidorgenase (IVDH) e (D)-2-hidroxidoglutarato desidrogenase (D2HGDH) foram caracterizadas como doadores de elétrons para o pool de ubiquinone através do sistema ETF/ETFQO a partir da degradação de BCAA e lisina, respectivamente. Na verdade, o catabolismo de BCAA mostra-se de uma importância fundamental para nutrir o ciclo do ácido tricarboxílico, principalmente, em situações de estresse enquanto lisina mostra uma estreia associação com o ciclo do ácido tricarboxílico sendo importante para fazer um elo da degradação de aminoácido com a geração de energia. A transferência de elétrons através da fdoacadeia transportadora de elétrons mitocondrial acopla a síntese de ATP a partir da regeneração de NADH e FADH 2 para fosforilar ADP a ATP. Contudo, o conhecimento com relação a organização do sistema de fosforilação oxidativa (OXPHOS) e sua via alternativa sob limitação energética permanece escasso. Assim, essa tese, a qual se concentra no funcionamento da respiração em um contexto que o ciclo do ácido tricarboxílico e via alternativa como doador de elétrons para cadeia transportadora de elétrons mitocondrial, é composta por três independentes capítulos centrados no metabolismo energético e respiração alternativa em Arabidopsis thaliana. Por isso, para se obter uma visão global de como ocorre o envolvimento e interação do ciclo do ácido tricarboxílico juntamente da via alternativa para coordenar o ajustamento das necessidades metabólicas e celulares, três abordagens experimentais foram usadas (i) uma abordagem in silico, nós investigamos a história evolucionária dos genes do ciclo do ácido tricarboxílico gerando um modelo para a origem dos genes do ciclo em plantas bem como seu comportamento submetido a uma série de estresse; (ii) a importância da biossíntese de lisina foi investigado usando mutante de Arabidopsis com reduzida atividade da enzima L,L-diaminopimelato aminotransferase (dapat) da via biossintética de lisina; (iii) reprogramação metabólica do sistema OXPHOS associado a limitação de carbono foi investigado. Rapidamente, os resultados apresentados aqui forneceram resultados que permitiu, no mínimo um prévio, a elaboração de mecanismo do metabolismo energético junto a vias alternativas. Primeiramente, permitiu a elucidação da origem evolutiva dos constituintes do ciclo do ácido tricarboxílico em plantas fornecendo elemento para a origem das isoformas presentes nos diferentes compartimentos subcelulares os quais que devem ser associados com eventos de transferência gênica ou com novas cópias geradas por duplicação genômica. Ademais, análises de co-expressão dos genes do ciclo em diferentes condições estressantes em ambos tecidos parte aérea e raiz demonstrou a presença de plasticidade molecular e forneceu uma explicação para o funcionamento do ciclo do ácido tricarboxílico em plantas. Após isso, o uso de Arabidopsis mutante com reduzida atividade para biossíntesi de lisina L,L-diaminopimelato aminotransferase (dapat) foi demonstrada que biossíntese de lisina simula condições de estresse e impacta no crescimento e metabolismo foliar. Por fim, uma avaliação de como o comportamento do sistema OXPHOS sob limitação de carbono e como vários aminoácidos podem impactar os complexos respiratórios foi possível demonstrar que o sistema OXPHOS tem sua função afetada por diferentes fontes de carbono e que vias alternativas são induzidas sob essas condições. Ademais, imunoensaios revelaram que é mais provável ser regulado por modificações pós traducionais. Juntos, esses resultados realçam a complexidade e especificidade da respiração vegetal durante evolução e que é differentemente afetado por linitações energéticas e pelo uso de substratos alternativos. Os resultados discutidos aqui suportam que ETF/ETFQo é uma via essencial capaz de doa elétrons para a cadeia transportadora de elétrons e que amioácidos são substratos alternativos para manter a respiração sob limitação de carbono. Os resultados obtidos são discutidos em um contexto de evolução metabólica mostrando estreia associação da metabolismo energético com metabolismo de aminoácidos e onde possível mcanísticos são devidamente discutidos. Palavras chaves: ciclo do ácido tricarboxílico; escassez de energia; evolução mitochondrial; fosforilação oxidativa; genes parálogos, metabolismo mitocondrial; neofuncionalização; respiração; resposta a estresse; substratos alternativos<br>Plant mitochondrion are involved in several key cellular processes that goesbeyond energy production being also associated with programmed cell death, fruit ripening and even light- associate process including phorespiration and photosynthesis. In this context, mitochondria acquisition by host cell brought evolutionary advances for the existing plant cell by the preservation of diverse metabolic pathways including both those related to energy metabolism as well as those associated with lipids, nucleotides and vitamin biosynthesis. The most notorious heritage is related to the tricarboxylic acid (TCA) cycle. The TCA cycle is an essential pathway which is related to reducing power (NADH and FADH2) generation, nitrogen assimilation and photosynthesis optimization. It has been suggested that the TCA cycle operated as isolated steps prior endosymbiosis events and that only after mitochondria acquisition it was possible for it to be organized and function as a cycle. The TCA cycle is composed by a set of eight enzymes. However, each enzyme is encoded by several genes which are targeting not just to mitochondria, but that are also imported into others subcellular compartments. These TCA enzymes located in other subcellular compartiments result in likely a broader connection between mitochondria and other organelles (e.g. peroxissome and chloroplast) allowing a bypass of the intermediates of the cycle switching his operation to an unusual in non-cyclic modes flux. It is also currently accepted that under stress conditions, which leads to decreases in carbohydrate levels, the TCA cycle can function in non-cyclic flux mode due to diminishing of carbon skeleton the enter it making required that be fed by anauplerotic reactions. Therefore, amino acids become essential to support respiration and ATP synthesis under such situations. Compelling evidence have demonstrated that branched chain amino acids (BCAA) and lysine can supply electrons to the mitochondrial electron transport chain (mETC) by the action of the electron transfer flavoprotein (ETF)-ETF: ubiquinone oxidoreductase (ETF/ETFQO) system and associated dehydrogenases. In plants, only isovaleryl- CoA dehydrogenase (IVDH) and (D)-2-hydroxyglutarate dehydrogenase (D2HGDH) have been characterized as electron donnor to the ubiquinol pool via this system so far by the degradation of BCAA and lysine, respectively. In fact, BCAA catabolism is of pivotal importance to provide intermediates to TCA cycle, particularly under stress situations, whereas lysine shows a strict association with the TCA cycle being required to couple amino acid degradation and energy generation. The electron transfer through the mETC is tightly coupled to ATP synthesis and use electron donates by NADH and FADH 2 to phosphorylate ADP to ATP. However, our knowledged regarding the organization of the mitochondrial oxidative phosphorylation (OXPHOS) system and its alternatives pathways under energy limitation remains elusive. Thus, this thesis, which is focused on the function of respiration within the context of the role of the TCA cycle as well as the function of alternative electron donors to the mETC, iscomprised by three independent stand-alone chapters focusing on energy metabolism and alternative respiration in Arabidopsis thaliana. Hence to obtain a compreenhesive picture of how the TCA cycle evolved and to which extend its alternative pathways interact to adjust to different cellular and metabolic requirements, three experimental approaches were used: (i) by using bioinformatic approaches we investigated the evolutionary history of TCA cycle genes allowing the generation of a model for the origin of the TCA cycle genes in plants and connected its evolution with TCA cycle behavior under a range of stress; (ii) the importance of lysine deficiency were investigated by using an Arabidopsis mutant with reduced activity of the lysine biosynthesis enzyme L,L-diaminopimelate aminotransferase (dapat), and (iii) the metabolic reprograming associated with the OXPHOS system were investigated following carbon limitation.. In brief, the results presented here provided several novel findings and allowed, at least preliminarly, mechanistic interpretation thereof. First, it facilitate the elucidation of the evolutionary origem of the TCA cycle in land plants providing support to the contention that the origin of isoforms present in different subcellular compartments might be associated either with gene-transfer events which did not result in correct targeting or with new gene copys generated by genome duplication and horizontal transfer gene. Additionally, coexpression analyses of TCA cycle genes following different stress conditions in both shoot and root tissues demonstrated the presence of a large molecular plasticity and provided an explanation for the modular operation of the TCA cycle in land plants. Secondly, by using an Arabidopsis mutant with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase (dapat) it was demonstrated that lysine biosynthesis deficiency mimics stress situation and impacts both plant growth and leaf metabolism.Thirdly, by evaluating OXPHOS system behavior following carbon starvation and how a range of amino acids can impact respiratory complexes it was possible to further demonstrate that OXPHOS is affected in function of the carbon source and that alternative pathways are induced under this condition.In addition, immunoblotting assays revealed that OXPHOS system is most likely regulated by posttranslational modification. When considered together these results highlight the complexity and specificity of plant respiration during evolution and that it is differently affected following energy limitation by the usage of alternative substrates. The results discussed here support the contention that ETF/ETFQO is an essential pathway able to donate electrons to the mETC and that amino acids are alternative substrates maintaining respiration under carbon starvation.The results obtained are discussed in the context of current models of metabolic evolution showing the strict association of energy metabolism with amino acids metabolism, and where possible, mechanistic insights are properly discussed. Key-words: alternative substrate respiration; energy deprivation; mitochondria evolution; mitochondria metabolism; neofunctionalization; OXPHOS; paralogous genes; stress response; TCA cycle;

Les troubles des conduites alimentaires (TCA) représentent aujourd'hui un problème majeur de santé publique dans la plupart des pays occidentaux. Les chercheurs ont mis en évidence la nécessité de mieux comprendre l?influence de la régulation émotionnelle sur les troubles des comportements alimentaires. Nous avons tenté d'étudier un large registre de processus émotionnels et leurs liens avec différentes dimensions psychopathologiques et neuropsychologiques. Ceci afin de mieux définir les différentes composantes qui sous-tendent les TCA, mieux comprendre la nature de leurs profils spécifiques, permettre ainsi de mieux les catégoriser. Poser ainsi les bases d?une catégorisation plus fine des troubles devrait permettre à terme d?améliorer la spécificité des prises en charge thérapeutiques et le pronostic. Notre population se compose de 23 anorexiques, 26 boulimiques comparées avec 41 sujets contrôles. Nous avons étudié la dysrégulation émotionnelle chez les patientes TCA à travers l?étude de traits émotionnels (alexithymie, anxiété, impulsivité, colère, labilité émotionnelle), et différentes mesures cognitives (attention, mémoire de travail, inhibition, flexibilité cognitive, prise de décision). Pour chacune de ces variables la recherche des ses corrélats avec les autres dimensions (alimentaires, émotionnelles et cognitives) nous a permis de mieux spécifier ce trouble. Notamment nos résultats mettent en évidence une dysrégulation émotionnelle non spécifique chez les patientes TCA<br>The eating disorders (ED) represent today a major problem of public health in the majority of the Western countries. The researchers highlighted the need for better understanding the influence of the emotional regulation on the food behavioral problems. We tried to study a broad register of emotional processes and their bonds with various psychopathological and neuropsychological dimensions. This in order to better define the various components which underlie the ED, to better understand the nature of their specific profiles, thus to allow to better categorize them. Thus to pose the bases of a finer categorization of the disorders should make it possible to improve the specificity of the responsibility of therapeutic assumptions and the forecast. Our population is composed of 23 anorexics, 26 bulimics compared with 41 subject controls. We studied the dysregulation emotional in patients ED through the study of emotional features (alexithymia, anxiety, impulsivity, anger, emotional lability), and various cognitive measurements (attention, working memory, inhibition, cognitive flexibility, decision-making). With a search of their relationship to other dimensions (food, cognitive and emotional) enabled us to better specify this disorder. In particular our results highlight a nonspecific dysregulation emotional in patients ED

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第9840号<br>農博第1300号<br>新制||農||855(附属図書館)<br>学位論文||H14||N3727(農学部図書室)<br>UT51-2003-B380<br>京都大学大学院農学研究科応用生命科学専攻<br>(主査)教授 島田 幹夫, 教授 加藤 暢夫, 教授 江﨑 信芳<br>学位規則第4条第1項該当

Dissertação para obtenção do Grau de Mestre em Engenharia Física<br>A contaminação do vinho que produz cheiro a “mofo” é um problema que afeta a indústria corticeira. O principal agente contaminante é 2,4,6-TCA que, em solução alcoólica, o limiar de deteção humana é da ordem de 5 ng/L. As características da rolha de cortiça tornam difícil a sua descontaminação e os métodos de deteção disponíveis atualmente não permitem a aplicação na indústria. Neste trabalho, é explorada a deteção de gases halogenados pela formação de uma dupla camada na superfície de um emissor de iões alcalinos. Os iões alcalinos são emitidos por ionização superficial. A corrente iónica alcalina em vácuo segue, nas condições experimentais, a lei de Child-Langmuir e a função de trabalho é calculada num determinado intervalo de temperatura pela equação de Saha-Langmuir. Embora os resultados tenham tido um sucesso limitado, verificou-se que a introdução de clorofórmio (como exemplo de gás halogenado) produziu um aumento da corrente iónica que depende da temperatura e da pressão. A recuperação da corrente foi feita ao aquecer o emissor até cerca de 1200K durante alguns minutos. O campo eléctrico de extração que foi aplicado variou entre 30V/cm e 135V/cm em regime contínuo e a temperatura variou entre 800K e 1200K.

La voie intrinsèque de la coagulation sanguine a été modélisée au moyen de 23 réactions élémentaires (13 activations enzymatiques, 2 complexations et 8 inhibitions), depuis l'activation du facteur XII jusqu'au caillot de fibrine. Grâce aux constantes cinétiques trouvées dans la littérature et optimisées par la méthode du simplex, le Temps de Céphaline + Activateur (TCA) a été simulé et comparé à des valeurs expérimentales. L'optimisation du modèle s'est appuyée sur les courbes d'étalonnage usuellement utilisées en laboratoire et sa validation sur l'analyse de plasma pathologique. Le modèle permet de discuter de la pertinence d'hypothèses émises antérieurement pour expliquer le mécanisme d'activation du facteur XI. La comparaison des résultats en milieu déficitaire en facteur XI ou XII confirme que l'hypothèse d'une activation du facteur XI par le facteur XIla seul ne peut être retenue. Ces mêmes résultats suggèrent la prédominance d'un mécanisme d'autoactivation du facteur XI plutôt qu'une médiation par la thrombine. De plus, la simulation permet de prévoir les profils de concentration en facteur VIlla, IXa, XIa, XIIa, prothrombine, thrombine et fibrine ainsi que la sensibilité du TCA face à la détection de déficits uniques (en facteur VIII, IX, XI ou XII) et combinés (en facteurs VIII et IX). L'effet de la présence initiale de traces de facteur XII a en quantité variable a aussi été abordé<br>The intrinsic pathway of blood coagulation was modelized using 23 elementary reactions including 13 enzymatic activations, 2 active complex formation and 8 inhibitions, which progress from activation of factor XII activation to formation of the fibrin clot. With the support of rate constant values found in literature and optimized by simplex method, the Activated Partial Thromboplastin Time (APTT) was simulated and compared to laboratory determinations, Standardization curves established routinely in the laboratory were used for the optimization while analysis of pathological plasma served to validate the mode!. This modelization allows us to discuss the pertinence of hypothesis drawn before to explain the rnechanism of factor XI activation. Comparative results obtained in factor XI- or factor XII- deficient plasma confirrned that factor XI cannat be solely activated by factor XIla. These results suggest predominant autoactivation of factor XI instead of a mediation by thrombin. In addition, the simulation predicts the time-course progress curves of factor VIlla, IXa, XIa, XIIa, prothrornbin, thrombin and fibrin, and the sensitivity of APTT to single (VIII, IX-, XI- or XII) or combined (VIII and IX) deficient factors. The effect to initial and varying trace amounts of factor XIIa was also examined

Contexte : Les personnes atteintes de troubles des conduites alimentaires (TCA) auraient un meilleur pronostic s’ils étaient pris en charge précocement pour cette maladie. Le médecin généraliste (MG) est souvent cité dans les recommandations internationales comme un acteur principal de dépistage et de prise en charge précoce des patients atteints de TCA. Les études de prévalence effectuées dans un contexte de soins primaires (SP) ne retrouvent pourtant que peu de patients atteints de TCA, voire aucun. Aucune étude de prévalence en MG en France n’a encore été effectuée. La dépression est souvent étudiée comme une comorbidité fréquente des TCA.Objectif : Etudier la place du MG dans la prise en charge des patients atteints de TCA1)Evaluer l’efficacité d’un dépistage systématique des patients atteints de TCA par des MG sur leur pronostic et leur taux de guérison.2)Décrire les caractéristiques des patients atteints de TCA et suivi en MG et d’étudier la temporalité entre la survenue d’une prise en charge pour dépression et prise en charge pour TCA3)Décrire les perceptions des MG et des patients de la place du MG dans la prise en charge de ces patientsMéthodes1)Revue systématique de la littérature selon les critères PRISMA2)Etude de cohorte à partir de la prise en charge en médecine générale de patients atteints de TCA avec analyse de différents groupes de suivi comparant les patients TCA pris en charge pour dépression et ceux non pris en charge pour dépression.3)Etude qualitative en miroir de patients atteints de TCA et de MG sélectionnés dans la cohorte disponible des MG, avec analyse par théorisation ancrée et phénoménologique en miroir, avec double codage d’entretiens semi directifsRésultats : La revue systématique de la littérature n’a pas permis de conclure à l’efficacité d’un dépistage systématique des TCA par les MG, du fait d’une littérature hétérogène, composée en grande majorité d’articles d’opinion dans un contexte de SP.1310 patients (sur 350 000 patients suivis) ont eu au moins une prise en charge pour TCA par des MG de l’OMG entre 1994 et 2007. 80 % étaient des femmes, âgées de 35,19 ans en moyenne. Seuls 39 % avaient une amorce de suivi pour TCA. 67,1 % avaient une première prise en charge pour TCA. 32,3 % d’entre eux avaient eu au moins une fois une prise en charge pour dépression, dont 62,41 % pour la première fois. Les patients ayant une dépression étaient plus âgés, plus suivis pour TCA que les autres. Un cinquième des patients TCA ont eu au moins une fois une prescription d’antidépresseurs dans leur suivi, un peu moins de la moitié des anxiolytiques. La prise en charge pour DEP ne précédait pas la prise en charge du TCA.Dans l’étude qualitative, les 24 MG et les 8 patientes décrivaient la prise en charge comme difficile et longue. L’enjeu d’une bonne prise en charge dépendait d’une relation de confiance des deux acteurs, d’un côté une patiente prête à se livrer en dépassant le déni et le sentiment de honte, de l’autre côté, un médecin prêt à écouter, en dépassant son sentiment d’impuissance.Discussion : Il s’agit de la première étude en France s’intéressant à la prise en charge des patients en SP, utilisant des méthodes complémentaires. Les enjeux de prise en charge des TCA en SP restent à définir. La dépression et les TCA étaient concomitants et semblent devoir être pris en charge dans une approche globale. Cette approche devra tenir compte de situations retrouvées dans les deux syndromes : la mauvaise estime de soi, une image négative de son corps, des antécédents de maltraitance, des antécédents familiaux psychiatriques de dépression ou de TCA au premier degré<br>Background : People suffering from eating disorders (ED) may have a better prognosis if they benefit from early management for their disease. GPs are often quoted in international guidelines as the main actors of screening and early management of ED patients. Prevalence studies in a primary care setting however find few ED patients and sometimes none. No study in GP has been done in France. Depressive disorders are often related as a comorbidity of ED. Aim: to study the GP role of ED patients management. 1) To evaluate the efficacy of systematic screening of ED patients conducted by GPs on prognosis and recovery. 2) To describe the characteristics of ED patients followed by their GPs and study the temporality between depression management and ED management. 3) To describe GPs and patients' views of the role of GPs management of ED patients. Methods 1)Systematic review according to PRISMA statement 2)Cohort study in a French GP setting of ED patients with analysis of different follow up groups comparing ED patients with depression to ED patients without depression. 3)Qualitative study of ED patients comparing GPs and patients' views selected in the cohort study, with a grounded theory approach and a phenomenological approach, double coding of semi directive interviews. Results : the systematic review found no evidence of efficacy of systematic screening of ED patients by GPs. The literature is too heterogeneous and contains mainly opinion papers in primary care. 1310 patients (out of 350,000) had at least one consultation for ED by GPs in the French database between 1994 and 2007. 80 % were women, mean age of 35.19 years. Only 39 % benefited from a follow up for ED. 67.1 % were taken in charge for the first time. 32.3 % of ED patients had at least one consultation for depression, 62.41 % for the first time. ED patients with depression were older, followed longer for ED then the others. One fifth of patients had at least one prescription of antidepressants during their follow up period, half of them had anxiolytics. Depression management did not precede ED management. In the qualitative study, 24 GPs and 8 patients described the management as difficult and long. The key to a good management depended on trust, with a patient willing to talk about her disease; overcoming shame and denial, and a GP ready to listen, overcoming his helplessness feeling.Discussion : it was the first study about this subject in France, using complementary methods. Management points are to be defined in primary care. Depression and ED are cofactors and should be managed in a global approach. This approach will have to take into account signs found in those two syndroms: low self esteem, negative body image, abuse background, pscyhiatric diseases in family (depression and ED) in first degree relatives

Les troubles du comportement alimentaire les plus courants sont l’anorexie mentale et la boulimie nerveuse, ils représentent un grave problème de santé publique avec d’importantes conséquences physiques et psychiques. Dans un contexte de médecine de précision émergente, de plus en plus de domaines font appel au phénotypage des patients afin de permettre une meilleure prise en charge et la personnalisation de l’offre de soin. Ces travaux de recherches ont eu pour préoccupation l’anorexie mentale et la boulimie nerveuse avec la définition de la place de l’imagerie cérébrale métabolique et morphologique dans leur parcours thérapeutique et pronostique. Objectifs : Analyser le lien entre l’activité sérotoninergique cérébrale et l’efficacité de la fluoxétine dans la boulimie nerveuse ; Analyser le lien entre la morphologie et la fonction de l’hypophyse dans l’anorexie mentale ; Création d’un atlas IRM de l’hypophyse. Nous avons utilisé la tomographie par émission de positrons au radio ligand [18F]MPPF pour l’analyse de l’activité sérotoninergique cérébrale dans la boulimie nerveuse, les corrélations avec la réponse à la fluoxétine ainsi que les analyses voxel à voxel ont été réalisé par SPM. Pour l’étude de la morphologie de l’hypophyse dans l’anorexie mentale, nous avons délimité manuellement des lobes antérieurs et postérieurs de la glande et réalisé des corrélations avec les hormones hypophysaires. À partir de délimitations de l’hypophyse chez des femmes témoins nous avons créé un atlas IRM optimisé grâce à une approche de « masquage de la fonction de coût des lésions » avec SPM. Ont été mis en évidence, une corrélation négative entre le BPND [18F]MPPF du noyau du raphé dorsal (avant fluoxétine) et la réponse à la fluoxétine dans la boulimie nerveuse ainsi que des différences significatives entre le BPND [18F]MPPF avant et après la fluoxétine dans les régions limbiques chez les répondeurs mais pas chez les non répondeurs. Dans l’anorexie mentale, une corrélation entre le volume de l’hypophyse antérieur et l’hormone de croissance a été retrouvé. L’atlas IRM de l’hypophyse global, antérieur et postérieur a donné une bonne concordance et corrélation entre avec les méthodes manuelles. L’imagerie cérébrale qu’elle soit métabolique ou morphologique a définitivement sa place dans le domaine des troubles du comportement alimentaire. Elle permet d’élucider des mystères qui entourent encore la physiopathologie de ces troubles, nos résultats nous permettent d’ailleurs de proposer l’activité du noyau du raphé dorsal comme biomarqueur de la réponse à la fluoxétine dans la boulimie nerveuse. Quant au volume hypophysaire il nous apparait comme un véritable marqueur de la sévérité de la maladie. Notre atlas de l’hypophyse vise à nous affranchir des délimitations manuelles permettant un gain de temps non négligeable dans le quotidien de la recherche clinique<br>The most common eating disorders are anorexia nervosa and bulimia nervosa; they represent a serious public health problem with significant physical and psychological consequences. In the context of emerging precision medicine, various fields are increasingly turning to patient phenotyping to improve treatment and personalize healthcare. This research has focused on bulimia nervosa and anorexia nervosa, aiming to define the role of metabolic and morphological brain imaging in their therapeutic and prognostic pathways. Aims : Analyze the link between cerebral serotonergic activity and the efficacy of fluoxetine in bulimia nervosa ; Analyze the relationship between the morphology and function of the pituitary gland in anorexia nervosa ; Creation of an MRI atlas of the pituitary gland. We used positron emission tomography with the radioligand [18F]MPPF to analyze cerebral serotonergic activity in bulimia nervosa, and correlations with the response to fluoxetine, as well as voxel-wise analyses, were performed using SPM. For the study of pituitary gland morphology in anorexia nervosa, we manually delineated anterior and posterior lobes of the gland and conducted correlations with pituitary hormones. From pituitary delineations in control women, we created an optimized MRI atlas using a "lesion cost masking" approach with SPM. We found a negative correlation between BPND [18F]MPPF in the dorsal raphe nucleus (before fluoxetine) and the response to fluoxetine in bulimia nervosa. Significant differences in BPND [18F]MPPF were also observed before and after fluoxetine treatment in limbic regions among responders but not in non-responders. In anorexia nervosa, a correlation was found between the volume of the anterior pituitary and growth hormone. The MRI atlas of the global, anterior, and posterior pituitary showed good concordance and correlation with manual delineations. In the field of eating disorders, both metabolic and morphological brain imaging undoubtedly play a significant role. It helps unravel mysteries still surrounding the pathophysiology of these disorders. Our results also suggest the dorsal raphe nucleus activity as a biomarker for fluoxetine response in bulimia nervosa. As for pituitary volume, it appears to be a good marker of disease severity. Our pituitary atlas aims to eliminate manual delineations, providing a significant time-saving advantage in clinical research

Serial passaging of primary human fibroblasts leads to the formation of non-dividing senescent cells by a process termed replicative senescence. This tissue culture-based methodology is currently used as a model system to determine the underlying mechanisms of in vivo cellular aging and tumor suppression. Senescence is regarded as an alternative pathway to apoptosis, where cells undergo multiple changes in metabolic and cellular signaling pathways in order to prevent proliferation but still maintain a metabolically-active cell. Whether or not this model accurately reflects in vivo processes is presently controversial; however, replicative senescence is currently the most applicable model through which one can investigate the underlying causes of human cellular aging in the context of controlled environmental stress over time. This work was directed at understanding the molecular processes involved in replicative senescence with specific emphasis on the role of the mitochondria. A series of experiments were performed to assess changes during the induction of replicative senescence under conditions of low (3%) and high (20%) oxygen levels. Measurements were made at the transcriptional, protein, and metabolite levels. Microscopy wasalso utilized to monitor changes in mitochondrial morphology and volume. While previous studies have evaluated specific pathways and/or products; this work combines a more complete metabolomic, genomic, proteomic, and morphological picture of cells undergoing senescence and oxidative stress. Considering the low cell population densities of primary adherent fibroblasts and the subsequent low concentrations of small polar metabolites involved in glycolysis and the TCA cycle, methodologies needed to be developed in order to optimize metabolite extraction and liquid chromatography-mass spectrometric analysis. Protein kinase and transcriptional microarrays were also performed in order to quantify the changes in activated/deactivated signaling cascades as well as gene expression and relate these findings to metabolomic data. Mitochondrial dynamics of cells at different age time points and under different oxygen conditions were also assessed including mitochondrial size, shape, membrane potential, and percent volume per cell volume using confocal microscopy. The results obtained not only confirm the major pathways involved in senescence (p53/p21, PTEN/p27, and RTK/Raf/MAPK) but also provide evidence at both the transcriptional and protein levels for additional senescence-associated pathways. The majority of the changes observed were related to pathways involved in cellular stress, cell cycle control, and the survival response. Metabolic data suggested a –pooling effect– of glycolysis and TCA precursor molecules due to attenuation in enzyme function; this theory was also supported by an observed up regulation of gene expression as a compensatory mechanism. Mitochondria exhibited changes in membrane potential as well as volume and percent volume per cell which suggested compensatory hypertrophy and/or attenuation of mitochondrial fission processes. When the aforementioned analyses are tied together, a “theoretical model of senescence” can be formulated and is characterized by increased metabolic protein and associated metabolite levels due to attenuation in their respective enzyme function, resulting in increases in expression of their associated genes as a compensatory mechanism.<br>Ph. D.

OBJETIVOS: El objetivo general de la tesis fue el de obtener información sobre la prevalencia, persistencia, incidencia y factores de riesgo de los trastornos de la conducta alimentaria (TCA) al inicio de la adolescencia de la población escolar de la ciudad de Tarragona.<br/>MATERIAL Y MÉTODOS: Se trata de una investigación epidemiológica longitudinal en dos etapas. En la primera etapa (curso escolar 2002-03) se avaluó una muestra inicial de 1.336 escolares (649 chicos y 687 chicas; edad media: 11,37 anys) con un diseño en doble fase. Se seleccionaron por medio de un test de cribado (ChEAT) 258 alumnos, que fueron estudiados de manera individual (141 como grupo de riesgo y 117 como controles) (fase de T1) y que fueron seguidos a los dos años (T2; n = 200) (113 sujetos del grupo de riesgo y 87 del grupo control). Se les hizo una entrevista psiquiátrica estructurada según criterios DSM-IV (Entrevista Diagnóstica para Niños y Adolescentes- EDNA), para obtener los diagnósticos de TCA en las dos etapas (EDNA-Niños y EDNA-Adolescentes). También se obtuvieron datos antropométricos, de conducta alimentaria, de psicopatología, de desarrollo puberal, de influencias socioculturales, de temperamento y personalidad y de satisfacción corporal.RESULTADOS Y CONCLUSIONES: En primer lugar se presentan resultados sobre las características psicométricas de la versión catalana y adaptada del ChEAT, realizada experimentalmente para este estudio (Sancho et al, 2005). Un 12.94% de los escolares en la primera fase puntuaron igual o por encima del punto de corte del ChEAT. La prevalencia estimada de diagnósticos de TCA fue de 3, 44% en el T1 y de 3, 81% en el T2. Los diagnósticos denominados parciales (que no cumplen todos los criterios de severidad para ser considerados anorexia o bulimia nerviosas) -TCANE y subclínicos- son los diagnósticos más frecuentes. Sólo se halló un caso de bulimia en el T2. Un 52,17% de los participantes persistió con cualquier TCA a lo largo de los 2 años. Las diferencias de sexo fueron significativas en el T2, en el cual las chicas mostraron una mayor incidencia y persistencia que los chicos. Los escolares que tenían un IMC más alto fueron los que presentaban una mayor persistencia de diagnósticos (Sancho et al. 2007). Los escolares con diagnósticos parciales tenían características de personalidad y de temperamento de riesgo, comparables a las halladas en la población clínica con TCA. Las mujeres (43%) mostraron una mayor estabilidad en la insatisfacción corporal frente a los varones (25.8%). El sobrepeso en la preadolescencia está relacionado con la insatisfacción corporal en los varones y en las mujeres. La insatisfacción previa fue un predictor de insatisfacción corporal en ambos sexos, pero hallamos otros factores de riesgo diferentes como sería la pubertad tardía en los varones y el hecho de estar en sobrepeso en la preadolescencia en las mujeres.OBJETIVOS: El objetivo general de la tesis fue el de obtener información sobre la prevalencia, persistencia, incidencia y factores de riesgo de los trastornos de la conducta alimentaria (TCA) al inicio de la adolescencia de la población escolar de la ciudad de Tarragona.MATERIAL Y MÉTODOS: Se trata de una investigación epidemiológica longitudinal en dos etapas. En la primera etapa (curso escolar 2002-03) se avaluó una muestra inicial de 1.336 escolares (649 chicos y 687 chicas; edad media: 11,37 anys) con un diseño en doble fase. Se seleccionaron por medio de un test de cribado (ChEAT) 258 alumnos, que fueron estudiados de manera individual (141 como grupo de riesgo y 117 como controles) (fase de T1) y que fueron seguidos a los dos años (T2; n = 200) (113 sujetos del grupo de riesgo y 87 del grupo control). Se les hizo una entrevista psiquiátrica estructurada según criterios DSM-IV (Entrevista Diagnóstica para Niños y Adolescentes- EDNA), para obtener los diagnósticos de TCA en las dos etapas (EDNA-Niños y EDNA-Adolescentes). También se obtuvieron datos antropométricos, de conducta alimentaria, de psicopatología, de desarrollo puberal, de influencias socioculturales, de temperamento y personalidad y de satisfacción corporal.RESULTADOS Y CONCLUSIONES: En primer lugar se presentan resultados sobre las características psicométricas de la versión catalana y adaptada del ChEAT, realizada experimentalmente para este estudio (Sancho et al, 2005). Un 12.94% de los escolares en la primera fase puntuaron igual o por encima del punto de corte del ChEAT. La prevalencia estimada de diagnósticos de TCA fue de 3, 44% en el T1 y de 3, 81% en el T2. Los diagnósticos denominados parciales (que no cumplen todos los criterios de severidad para ser considerados anorexia o bulimia nerviosas) -TCANE y subclínicos- son los diagnósticos más frecuentes. Sólo se halló un caso de bulimia en el T2. Un 52,17% de los participantes persistió con cualquier TCA a lo largo de los 2 años. Las diferencias de sexo fueron significativas en el T2, en el cual las chicas mostraron una mayor incidencia y persistencia que los chicos. Los escolares que tenían un IMC más alto fueron los que presentaban una mayor persistencia de diagnósticos (Sancho et al. 2007). Los escolares con diagnósticos parciales tenían características de personalidad y de temperamento de riesgo, comparables a las halladas en la población clínica con TCA. Las mujeres (43%) mostraron una mayor estabilidad en la insatisfacción corporal frente a los varones (25.8%). El sobrepeso en la preadolescencia está relacionado con la insatisfacción corporal en los varones y en las mujeres. La insatisfacción previa fue un predictor de insatisfacción corporal en ambos sexos, pero hallamos otros factores de riesgo diferentes como sería la pubertad tardía en los varones y el hecho de estar en sobrepeso en la preadolescencia en las mujeres.OBJETIVOS: El objetivo general de la tesis fue el de obtener información sobre la prevalencia, persistencia, incidencia y factores de riesgo de los trastornos de la conducta alimentaria (TCA) al inicio de la adolescencia de la población escolar de la ciudad de Tarragona.MATERIAL Y MÉTODOS: Se trata de una investigación epidemiológica longitudinal en dos etapas. En la primera etapa (curso escolar 2002-03) se avaluó una muestra inicial de 1.336 escolares (649 chicos y 687 chicas; edad media: 11,37 anys) con un diseño en doble fase. Se seleccionaron por medio de un test de cribado (ChEAT) 258 alumnos, que fueron estudiados de manera individual (141 como grupo de riesgo y 117 como controles) (fase de T1) y que fueron seguidos a los dos años (T2; n = 200) (113 sujetos del grupo de riesgo y 87 del grupo control). Se les hizo una entrevista psiquiátrica estructurada según criterios DSM-IV (Entrevista Diagnóstica para Niños y Adolescentes- EDNA), para obtener los diagnósticos de TCA en las dos etapas (EDNA-Niños y EDNA-Adolescentes). También se obtuvieron datos antropométricos, de conducta alimentaria, de psicopatología, de desarrollo puberal, de influencias socioculturales, de temperamento y personalidad y de satisfacción corporal.RESULTADOS Y CONCLUSIONES: En primer lugar se presentan resultados sobre las características psicométricas de la versión catalana y adaptada del ChEAT, realizada experimentalmente para este estudio (Sancho et al, 2005). Un 12.94% de los escolares en la primera fase puntuaron igual o por encima del punto de corte del ChEAT. La prevalencia estimada de diagnósticos de TCA fue de 3, 44% en el T1 y de 3, 81% en el T2. Los diagnósticos denominados parciales (que no cumplen todos los criterios de severidad para ser considerados anorexia o bulimia nerviosas) -TCANE y subclínicos- son los diagnósticos más frecuentes. Sólo se halló un caso de bulimia en el T2. Un 52,17% de los participantes persistió con cualquier TCA a lo largo de los 2 años. Las diferencias de sexo fueron significativas en el T2, en el cual las chicas mostraron una mayor incidencia y persistencia que los chicos. Los escolares que tenían un IMC más alto fueron los que presentaban una mayor persistencia de diagnósticos (Sancho et al. 2007). Los escolares con diagnósticos parciales tenían características de personalidad y de temperamento de riesgo, comparables a las halladas en la población clínica con TCA. Las mujeres (43%) mostraron una mayor estabilidad en la insatisfacción corporal frente a los varones (25.8%). El sobrepeso en la preadolescencia está relacionado con la insatisfacción corporal en los varones y en las mujeres. La insatisfacción previa fue un predictor de insatisfacción corporal en ambos sexos, pero hallamos otros factores de riesgo diferentes como sería la pubertad tardía en los varones y el hecho de estar en sobrepeso en la preadolescencia en las mujeres.<br>OBJECTIVES: The aim of this study was to know the prevalence, to evaluate the persistence, the incidence and the risk factors of any eating disorders (ED) in a representative school population of early adolescents of both sexes in the city of Tarragona (Spain).<br/>MATERIAL AND METHOD: It is an epidemiological longitudinal study in two phases. In the first phase an initial sample of 1336 (649 boys and 687 girls, mean age: 11.37) were assessed in a two-phase design.258 subjects were selected with a screening test (ChEAT) from the initial sample and were assessed individually (141 as risk group and 117 as controls) (phase of T1), and were followed-up two years later (T2), n = 200, (113 from risk subjects and 87 from controls). <br/>Subjects were evaluated with a structured psychiatric interview according to DSM-IV criteria (Diagnostic Interview for children and adolescents DICA) to obtain diagnoses of ED using DICA-Children and DICA-Adolescents, at T1 and at T2 respectively. We also obtained data about anthropometry, eating behaviors, psychopathology, pubertal development, sociocultural influences, temperament and personality and body satisfaction.<br/>RESULTS AND CONCLUSIONS: First results are presented on the anthropometric characteristics of the adapted, Catalan version of Cheat, produced experimentally in this study (Sancho et al, 2005). 12.94% of schoolchildren in the first phase were rated equal to or above the cutoff point of the Cheat. The estimated prevalence of diagnoses of ED was 3, 44% at T1 and 3.81% at T2. Partial diagnoses (which does not meet all the criteria of severity to be considered anorexia or bulimia nervosa) -EDNOS and subclinic ED- are the most frequent diagnoses. Only there was a case of bulimia at T2.<br/>52.17% of the participants persisted with any ED at the follow-up two years later. Gender differences were significant at T2, being ED in females more prevalent, incident and persistent than boys. Participants who had the highest BMI were those who had a persistent diagnosis of some form of ED (Sancho et al. 2007). Subjects with non full-blown syndromes of eating disorders share characteristics such as temperament and character traits with subjects with full-blown syndromes. Girls showed more stability (43%) in body dissatisfaction than boys (25.8%). Overweight at preadolescence is related to body dissatisfaction in boys and girls. Dissatisfaction at preadolescence is an important predictor for body dissatisfaction in both sexes, but we found that there were other risk factors like late puberty in boys and overweight at preadolescence in girls.

Bacterial regulatory small RNAs (sRNAs) act as crucial regulators in central carbon metabolism by modulating translation initiation and/or degradation of target mRNAs in metabolic pathways. This work demonstrates that a noncoding sRNA, SdhX, is produced by RNase E-dependent processing from the 3 ́ untranslated region (3 ́ UTR) of an operon encoding three enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA (encoding an enzyme critical for degradation of the signaling molecule acetyl phosphate) without affecting the downstream pta gene that encodes the enzyme critical for acetyl phosphate synthesis. SdhX abundance is tightly coupled to the transcription signals of the TCA cycle genes but escapes all known post-transcriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways.<br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished

Mestrado em Bioquímica<br>A cortiça é um material de excelência e dos produtos com maior destaque na economia portuguesa, quer pelo elevado nível de exportações quer pela criação de emprego associada a este setor. A aplicação dada à cortiça com maior relevância económica é a rolha natural para vedação de vinhos. Contudo, alguns compostos halogenados presentes na cortiça, ao migrarem para o vinho, podem comprometer a sua qualidade levando a perdas significativas tanto para a indústria corticeira como para os produtores de vinho. Neste sentido, é de extrema importância o desenvolvimento e a aplicação de métodos de análise que permitam a quantificação simultânea das principais moléculas identificadas como responsáveis pelos desvios organoléticos dos vinhos. O presente trabalho surge com o objetivo de validar um método para análise simultânea de 2,4,6-tricloroanisol (TCA) e clorofenóis em cortiça e a sua posterior aplicação na análise de cortiças provenientes de diferentes locais da Península Ibérica, de forma a verificar uma possível relação entre a concentração destes compostos e as características edafoclimáticas de origem da cortiça. Com o objetivo de validar o método de análise, foram realizados estudos de linearidade, limiares analíticos, precisão, exatidão e robustez. Estes estudos permitiram considerar o método analítico como validado e implementá-lo nas análises de rotina do laboratório. Ao longo deste trabalho analisaram-se 46 amostras de cortiça, das quais 32 eram provenientes de Portugal e 14 de Espanha. Os resultados obtidos permitiram concluir que a cortiça da região da Andaluzia é aquela com valores médios e máximos mais elevados tanto para o TCA como para os clorofenóis, sendo a densidade do montado uma possível explicação para esta ocorrência, uma vez que, nesta região, o montado de sobro é muito denso (elevado número de árvores, pouca luz e exposição solar e mau arejamento), possivelmente propiciando o desenvolvimento fúngico e consequente formação destes compostos.<br>Cork is an excellence material and one of the most relevant products of the portuguese economy, either by the high level of exports and the number of jobs created by this industrial sector. The most important application of cork is as cork stopper for sealing wine bottles. However, some halogen containing compounds, by migrating to the wine, can compromise its quality causing significant financial losses for both the cork industry and winemakers. In this way, it’s extremely important the development and application of methods of analysis to allow the quantification of those molecules responsible for the sensory deviations of wine. This work arises with the aim of validating a method for the simultaneous analysis of 2,4,6-trichloroanisole (TCA) and chlorophenols in cork and it’s subsequent application in the analysis of cork samples from different locations in the Iberian Peninsula, to verify a possible relationship between the concentration of these compounds and the characteristics of the climate and the soil of the original cork tree. In order to validate the analytical method, linearity, analytical precision, accuracy and robustness studies have been performed. These studies allowed the validation of the analytical method and its implementation in the laboratory routine analysis. Throughout this thesis 46 cork samples were analyzed; 32 of them came from Portugal and 14 from Spain. The obtained results allow to conclude that the cork from Andalusian region is the one with higher values for both TCA and chlorophenols, being the density of the cork oak forest one possible explanation for this manifestation, once in this region the forest is denser (high number or trees, few light and sun exposure and bad aeration) providing, possibly, a higher fungi development and consequently the formation of these compounds.

This article presents a study conducted on data containing drug concentrations. The data was obtained from femoral venous blood samples collected at medico legal autopsies in Sweden. Cases positive for antidepressant drugs were scrutinized and divided in to two groups for 15 antidepressant drugs: B‑cases, where the cause of death was intoxication with more than one drug detected in the blood sample. C‑cases, where the cause of death was NOT intoxication and at least one drug (the antidepressant) was detected in the blood sample. This data was then processed to find frequencies of concomitant drugs taken together with the antidepressant drugs. Frequencies of the most common concomitant drugs were then compared between B-cases and C-cases for each antidepressant drug. This revealed that the drugs dextropropoxyphene, ethanol, codeine, flunitrazepam, paracetamol, propiomazine and alimemazine were signifcantly more common as concomitant drugs in B-cases (intoxications) than in C‑cases (non‑intoxications). With regards to unknown interactions the most interesting combinations were: Propiomazine with mirtazapine, venlafaxine, citalopram or fluoxetine; Paracetamol with paroxetine; Flunitrazepam with mirtazapine, venlafaxine or citalopram; Codeine with mirtazapine or sertraline. These combinations should be further investigated.

Le présent travail porte sur l'étude du traitement par Détente Instantanée Contrôlée (DIC) de l'élimination du TCA et des autres molécules responsables du "goût de bouchon", des granulés de liège en vue de l'amélioration et la maîtrise de sa qualité. L'analyse chromatographique par SPME-HS a prouvé l'importance de l'étape d'humidification avant le traitement et le séchage après le traitement dans l'élimination du TCA. L'effet des paramètres opératoires (pression de vapeur, temps de traitement et temps de vide final) a été quantifié. Le traitement DIC a permis de réduire le taux du TCA extractible résiduel à moins de 0. 2ng/L. L'efficacité d'élimination du TCA est 100% dans les granulés (0. 5 - 1mm), (2 - 4mm) et (4 - 7mm). L'effet du traitement DIC sur la cinétique de séchage des granulés de liège a été étudié. En général, ce traitement augmente légèrement la vitesse moyenne de séchage. Les valeurs de la diffusivité effective du liège ont varié entre 2. 63 x 10-10 à 4. 49 x 10-9 m2. S-1 pour les échantillons non traités alors qu'elles varient de 2. 27 x 10-10 à 6. 00 x 10-9 m2. S-1 pour les échantillons traités, dans la gamme de température de 50° à 70°C. Les valeurs d'énergie d'activation des granulés de liège sont respectivement de 25. 29 à 48. 12 kJ/mol et de 32. 43 à 43. 98 kJ/mol pour l'échantillon non traité et traité par DIC. La Déshydratation par Détentes Successives (DDS) a été étudiée. Les isothermes d'adsorption des granulés de liège sont déterminés à 25, 40 et 60°C et ajustées selon les modèles de GAB, BET et Henderson<br>The present work concerns the study of Instant Controlled Pressure-drop (DIC) treatment on the elimination of the TCA and other molecules which responsible of the cork taint from the cork granules in order to control its quality. The chromatographic analysis by SPME-HS proved the importance of the humidification stage before treatment and drying after the treatment in elimination of the TCA. The effect of the DIC operating parameters (steam pressure, treatment time and final vacuum time) was quantified. The DIC treament was able to reduce the rate of residual extractable TCA to less than 0. 2ng/L. The effectiveness of elimination of the TCA was 100% for the cork granules of different diameters (0. 5 - 1, 2 - 4 and 4 - 7 mm). The effect of treatment DIC on the drying kinetics of cork granules was studied. In general, the DIC treatment slightly increased the drying rate. The values of the effective diffusivity of cork granules varied between 2. 63 x 10-10 to 4. 49 x 10-9 m2. S-1 for the untreated samples whereas for the DIC treated samples its varied between 2. 27 x 10-10 to 6. 00 x 10-9 m2. S-1 in the range of 50° - 70°C. Values of the activation energy were ranged between 25. 29 to 48. 12 kJ/mol and 32. 43 to 43. 98 kJJ/mol for untreated and DIC treated cork granules respectively. Dehydration by Successive Pressure Drop (DDS) was studied for cork granules (0. 5 - 1mm). The adsorption isotherms of cork granules was determined at 25, 40 and 60°C and adjusted according to the models of GAB, BET and Henderson

La thérapie de pleine conscience est considérée comme la nouvelle orientation de la thérapie cognitive comportementale. Elle trouve son origine principalement avec le travail de Kabat-Zinn (1979- 2009), le président du centre CFM : Centre for Mindefulness in médecine, qui a montré que la thérapie de pleine conscience est une thérapie autonome capable de diminuer des symptômes pathologiques et influencer la personnalité pour aboutir à des changements cognitifs parfonds. Dans notre travail l’anorexie mentale a été choisie comme une pathologie pouvant être traitée par cette thérapie, ce qui n’avait pas été effectué jusqu’à présent. Les séances se sont déroulées avec des patientes hospitalisées à Lyon et à St. Etienne. Les résultats obtenus ont montré que la prise en charge par « la pleine consciences », a fait diminué les symptômes pathologiques d’anorexie mental (physique, psychique et cognitifs), et que les patientes, après la thérapie, ont présenté un locus de contrôle interne par rapport au début de thérapie et par rapport au groupe contrôle ; qu’elles utilisent moins le coping émotion et plus le coping problème et le coping soutien social par rapport au début la thérapie et par rapport au groupe de contrôle<br>The Mindfulness therapy is considered the new direction of cognitive behavioral therapy. It started mainly with the work of Kabat- Zinn (1979 - 2009) President of CFM Centre: Centre for Medicine in Mindefulness where he interested in managing stress using mindfulness. This study is an attempt to make the therapy of mindfulness as an independent therapy by itself is able to reduce disease symptoms and influence on personality to cognitive changes Profound. It targets to show that this type of therapy is far from being a simple tool to manage stress. Anorexia nervosa has been selected as pathology to be treated by this therapy, which held its session in patient hospitalized in Lyon and St. Etienne. The results showed that the treatment with the Mindfulness had reduced the pathological symptoms of anorexia nervosa (physical, emotional and cognitive), and that patients, after therapy, had became with a locus of control internal Compared with the beginning of treatment, and with the control group, they used less the coping emotional and coping problem and coping social support compared with the start of therapy and the control group

La survie des cellules cancéreuses dépend d’une activité énergétique et biosynthétique accrue et la glutamine participe à de nombreux processus nécessaires à cette adaptation métabolique. Dans les leucémies aiguës myéloïdes (LAM), la croissance et la prolifération sont favorisées par l’activation anormale de plusieurs voies de signalisation, et notamment par la voie mTORC1. Les acides aminés essentiels, et en particulier la leucine, sont indispensables à l’activation de mTORC1. La glutamine est captée par la cellule via le transporteur SLC1A5 et permet ensuite l’entrée de la leucine via le transporteur bidirectionnel SLC7A5. La concentration en glutamine est donc une étape limitante dans l’activation de mTORC1 par la leucine. Nous avons étudié les effets de la privation en glutamine dans les LAM à l’aide de différents outils (milieu sans glutamine, shARN inhibant l’expression du transporteur de la glutamine SLC1A5 et la drogue L-asparaginase, qui a une activité de glutaminase extracellulaire), et observé une inhibition de mTORC1 et de la synthèse protéique. L’inhibition du transporteur SLC1A5 inhibe la pousse tumorale dans un modèle de xénotransplantation. La L-asparaginase inhibe mTORC1 et induit une apoptose de façon proportionnelle à son activité glutaminase et complètement indépendante de la concentration en asparagine. La privation en glutamine induit l’expression de la glutamine synthase et l’autophagie, et ces deux processus peuvent être des mécanismes de résistance intrinsèques ou acquis dans certaines lignées leucémiques. L’apoptose induite par la privation en glutamine n’est cependant pas liée à l’inhibition de mTORC1, puisqu’elle n’est pas diminuée par l’utilisation d’un mutant de mTOR non inhibé par la privation en glutamine. Nous nous sommes donc intéressés à une autre voie dépendante de la glutamine dans de nombreux cancers, la phosphorylation oxydative. L’étape initiale du catabolisme intracellulaire de la glutamine est la conversion de la glutamine en glutamate par des enzymes appelées glutaminases. Différentes isoformes des glutaminases existent qui sont codées chez l’homme par les gènes GLS1 et GLS2. Le glutamate est ensuite transformé en α-cétoglutarate, intermédiaire du cycle TCA. Dans les lignées de LAM, la privation en glutamine inhibe la phosphorylation oxydative mitochondriale. Nous avons observé que la protéine glutaminase C (GAC), une des isoformes de GLS1, est constamment exprimée dans les LAM mais aussi dans les progéniteurs hématopoïétiques CD34+ normaux. L’inhibition d’expression de la GLS1 par des shARN inductibles ou bien par le composé CB-839 réduit la phosphorylation oxydative, conduisant à une inhibition de prolifération et à une induction d’apoptose des cellules leucémiques. L’invalidation génétique de la GLS1 inhibe la formation de tumeur et améliore la survie des souris dans un modèle de xénotransplantation. A l’inverse, le ciblage de la GLS1 n’a pas d’effets cytotoxiques ni cytostatiques sur les progéniteurs hématopoïétiques normaux. Ces effets anti-leucémiques sont inhibés par l’adjonction d’α-cétoglutarate, et ceux induit par le CB-839 sont abrogés lorsqu’est exprimé de façon ectopique un mutant GACK320A hyperactif, attestant du rôle essentiel du maintien d’un cycle TCA actif dans les cellules de LAM. Enfin, nous montrons que l’inhibition de la glutaminolyse active la voie d’apoptose mitochondriale intrinsèque et agit en synergie avec l’inhibition spécifique de BCL-2 par l’ABT-199. Ces résultats démontrent que le ciblage spécifique de la glutaminolyse est une autre façon d’exploiter l’addiction à la glutamine des cellules leucémiques de LAM et que le maintien d’un cycle TCA actif est essentiel à la survie de ces cellules<br>Cancer cells survival is dependent on high energetic and biosynthetic activity, and glutamine is involved in many metabolic processes necessary for this adaptation. In acute myeloid leukemia (AML), growth and proliferation are promoted by activation of several signaling pathways, including mTORC1. Essential amino acids, in particular leucine, are required for mTORC1 activation. Glutamine enters into the cell via the SLC1A5 transporter and then allows the input of leucine via the bidirectional SLC7A5 transporter. Therefore, the intracellular glutamine concentration is a limiting step in the activation of mTORC1 by leucine. We studied the effects of glutamine deprivation in AML using different tools (medium without glutamine, shRNA against the SLC1A5 glutamine transporter and the drug L-asparaginase, which has an extracellular glutaminase activity) and observed mTORC1 and protein synthesis inhibition. SLC1A5 transporter knockdown inhibits tumor growth in a xenotransplantation model. L-asparaginase inhibits mTORC1 and induces apoptosis in proportion to its glutaminase activity and independently of asparagine concentration. Glutamine privation induces the expression of glutamine synthase and autophagy, and these two processes are involved in the resistance to glutamine privation in some leukemic cell lines. However, apoptosis induced by glutamine privation is not related to the inhibition of mTORC1, since it is not modified in the presence of a constitutively active mutant of mTOR. We next focused on the oxidative phosphorylation, another glutamine dependent pathway in many cancers. The initial step of the intracellular catabolism of glutamine is the conversion of glutamine to glutamate by enzymes called glutaminases. Different glutaminases isoforms exist that are encoded by the GLS1 and GLS2 genes. Glutamate is then converted to α-ketoglutarate, an essential TCA cycle intermediate. In AML cell lines, we observed that glutamine privation inhibits mitochondrial oxidative phosphorylation. The protein glutaminase C (GAC), an isoform of GLS1, is constantly expressed in AML but also in normal CD34 + hematopoietic progenitors. The knockdown of GLS1 by inducible shRNA or by the CB-839 compound reduced oxidative phosphorylation, leading to proliferation inhibition and apoptosis induction in leukemia cells. Genetic invalidation of GLS1 inhibits tumor formation and improves survival of mice in a xenograft model. Conversely, the targeting of GLS1 has no cytotoxic or cytostatic effects on normal hematopoietic progenitors. These anti-leukemic effects are inhibited by the addition of α-ketoglutarate, and those induced by the CB-839 are suppressed in the presence of an ectopically expressed GACK320A hyperactive mutant, confirming the essential role of maintaining an active TCA cycle in AML cells. Finally, we showed that glutaminolysis inhibition induces the intrinsic mitochondrial pathway of apoptosis and acts synergistically with the specific inhibition of BCL-2 by ABT-199. These results demonstrate that specific targeting of glutaminolysis is another way to exploit glutamine addiction in AML and that an active TCA cycle in essential for AML cell survival

INTRODUCTION: A dysfunction in fatty acid beta-oxidation (β-oxidation), particularly medium chain acyl-CoA dehydrogenase (MCAD) dysfunction is a major cause of mortality and its diagnosis is usually achieved by measuring specific protein activities or metabolites in blood and/or urine samples. However, these methods do not account for secondary defects that accompany primary deficiency; such as where measures of disruption in fatty acid metabolism do not account for defects in the TCA cycle and oxidative phosphorylation. These metabolic pathways are connected and dysfunction in one pathway (primary) could lead to dysfunction in the other (secondary). We propose the use of methods that combines all aspects of the bioenergetics module (enzyme activity in substrate oxidation within each individual pathway, transfer of electrons through the electron transport system (ETS) and oxidative phosphorylation for ATP generation) may be a more effective assessment technique. High resolution respirometry (HRR) is a recently developed technique that accounts for substrate oxidation, electron transfer via the ETS and oxidative phosphorylation. It measures the rate of oxygen consumption or flux at different respiratory states when appropriate substrates, uncouplers and inhibitors (SUIT protocols) are used. With this method, two substrate combinations are commonly used to assess medium-chain fatty acid β-oxidation; a) Octanoylcarnitine and carnitine, which is partial to the β-oxidation cycle alone, and b) Octanoylcarnitine and malate, which assesses the influence of the TCA cycle. Additionally, a combination of pyruvate, glutamate and malate is used to assess oxidation within the TCA cycle. We investigated the sensitivity of commonly used substrate combinations in HRR assessment to detect changes in mitochondrial respiration and dysfunction induced by the inhibition of either β-oxidation or the TCA cycle in C2C12 myotubes. Furthermore, we assessed MCAD, citrate synthase and aconitase enzyme activities when β-oxidation or TCA cycle was inhibited in C2C12 myotubes. METHODS: C2C12 myotubes were differentiated for 6 days and treated for 12 hours with a high or a low concentration of one of two inhibitors as follows; a) 2 mM or 8 mM 2-mercaptoacetate to inhibit medium chain acyl-CoA dehydrogenase (MCAD); b) 6 mM or 9 mM fluorocitrate to inhibit aconitase. Each treatment was compared to control myotubes grown for the same length of time without the addition of inhibitors. The activities of MCAD, aconitase and citrate synthase were determined. In addition, mitochondrial respiration measured as O2 flux at Routine, Leak, OXPHOS and ETS respiratory states were assessed in an Oxygraph-2K after inhibition or in control treatments using; i) Octanoylcarnitine and carnitine ii) Octanoylcarnitine and malate iii) pyruvate, malate and glutamate substrate combinations. For each assessment we corrected O2 flux recorded at each state to; a) approximate number of cells (pmol O2/s/million cells) b) protein concentration (pmol O2/s/mg protein) c) Flux control ratio (FCR) of each state to the maximum ETS capacity; ETSFAO+CI+CII (convergent electron flow from Fatty acid oxidation (FAO), Complex I (CI) and CII) d) FCR to either FAO-linked ETS capacity; (ETSFAO) or CI-linked ETS capacity (ETSCI). RESULTS: Treatment of cells with either a low or high concentration of 2-mercaptoacetate to inhibit MCAD resulted in no significant difference in MCAD activity. Fluorocitrate treatment decreased aconitase activity with low treatment (p = 0.011) compared to control, and conversely it increased MCAD activity in high treatment compared to control (p = 0.024). Both 2-mercaptoacetate (p = 0.03) and fluorocitrate (p < 0.01) treatment at high concentrations resulted in increased citrate synthase activity, compared to low concentration and control. Mitochondrial respiration with octanoylcarnitine and carnitine substrate combination was not altered with MCAD or aconitase inhibition. Octanoylcarnitine and malate substrate combination showed a decrease in mitochondrial respiration at the following respiratory states with both MCAD and aconitase inhibition; Routine (p = 0.01), LeakFAO (p = 0.029), OXPHOSFAO (p = 0.006), ETSFAO (p = 0.008), ETSFAO+CI (p = 0.017). FCR of each state to the maximum capacity (ETSFAO+CI+CII) revealed a decrease with both MCAD and aconitase inhibition at the following states; routine (p = 0.001), OXPHOSFAO (p = 0.003), ETSFAO (p = 0.018), ETSFAO+CAR (p = 0.008) and ETSFAO+CI (p = 0.027). Pyruvate, malate and glutamate substrate combination showed decreased mitochondrial respiration with MCAD inhibition at the following respiratory states; Routine (p = 0.004), LeakCI (p = 0.007), OXPHOSCI (p = 0.003), ETSCI (p = 0.003), ETSCI+FAO (p = 0.01) and ETSCI+FAO+CII (p = 0.003). FCR of each state to the maximum capacity (ETSCI+FAO+CII) decreased with both MCAD and aconitase inhibition at Routine (p = 0.024), OXPHOSCI (p = 0.024) and ETSCI (p = 0.035) states. DISCUSSION: The main finding of this study was related to two of the SUIT protocols 1) octanoylcarnitine and malate, and 2) pyruvate, malate and glutamate. These protocols were sensitive in showing decreased respiratory capacity and coupling control ratios and may be appropriate for assessing changes in oxidative metabolism when there is a defect in β-oxidation and/ or the TCA cycle. On the other hand, octanoylcarnitine and carnitine substrate combination is not sensitive to detect dysfunction induced by inhibition of either β-oxidation or TCA cycle. Irrespective of the enzyme inhibited, HRR detected dysfunction in complex I (CI), although, when aconitase was inhibited, reduced CI-linked respiration was more pronounced compared to MCAD inhibition. Furthermore, primary inhibition of MCAD to inhibit β-oxidation may have caused secondary inhibition of TCA cycle via aconitase, shown in decreased TCA cycle CI-linked respiration where MCAD was inhibited. In contrast, primary inhibition of aconitase seemed to be compensated for by increased MCAD activity and mitochondrial respiration related to β-oxidation. Lastly, enzyme assaysshould not be used as standalone techniques for assessing metabolic dysfunction at the level of TCA, β-oxidation and the mitochondria since they are not sensitive to low level defects, nor do they account for secondary interactions that influence either TCA or betaoxidation. HRR is useful to assess mitochondrial respiration and dysfunction, when using an appropriate substrate combination and should be used in combination with the more traditional enzyme activity assays.

OBJETIVOS:<br/><br/>- Estimar la prevalencia de trastornos de la conducta alimentaria (TCA) según diferentes niveles de severidad (diagnóstico de anorexia y bulimia nerviosa, de trastornos de la conducta alimentaria no especificados (TCANE) y síntomas de TCA) en adolescentes del municipio de Tarragona y de zona rural de la misma provincia.<br/>- Describir y analizar según los diferentes niveles de severidad de alteración de la conducta alimentaria.<br/>- Valorar la influencia de los diferentes factores sobre el riesgo de desarrollo de TCA.<br/><br/>MATERIAL Y MÉTODOS:<br/><br/>Estudio epidemiológico transversal en dos fases con adolescentes de ambos sexos que cursaban 1º y 3º de ESO (12-16 años) en 26 centros públicos y privados de la provincia de Tarragona. Primera fase (n=2967): se valoró la prevalencia de síntomas de alteración de la conducta alimentaria mediante dos test de cribado: Eating Attitudes Test (EAT) y Youth´s Inventory. Segunda fase: se estudió sólo el sexo femenino (n=295). Con los resultados de los test se seleccionaron dos grupos de individuos en función de la presencia (EAT>25 y Youth´s (+) para TCA) o ausencia de riesgo (controles) de TCA; valorandose: satisfacción y autopercepción corporal, influencia sociocultural, clima familiar, síntomas psicopatológicos, actividad física, conocimientos, preferencias y hábitos alimentarios mediante cuestionarios. Se valoró individualmente: el consumo de alimentos mediante el recordatorio de 24 horas, la composición corporal y se realizó la entrevista "Schedules for Clinical Assessment in Neuropsychiatry" (SCAN) en versión DSM-IV, CIE-10 para la obtención de diagnósticos.<br/><br/>RESULTADOS:<br/>El 13,3-17,1% de los participantes fueron considerados a riesgo de padecer un TCA (18,9-22,5%) mujeres, (7,6-11,7%) hombres dependiendo del test de cribado considerado. El nivel educativo de los padres es más bajo en las adolescentes con síntomas de TCA. Las situaciones familiares estresantes se asocian a una mayor prevalencia de síntomas de TCA. <br/>La prevalencia estimada de TCA en el sexo femenino es de 5,2% (IC 95%: 5-5,4), de anorexia nerviosa (0,53%) (IC 95%: 0,5-0,55), bulimia (0,66%) (IC 95%: 0,63-0-68) y TCANE (4 %) (IC 95%: 3,83-4,17 ). El nivel socioeconómico y educativo de los padres no esta significativamente asociado con TCA. En las adolescentes con TCANE se producen más situaciones familiares estresantes: divorcio padres, convivir con la nueva pareja del progenitor y/o con otras personas ajenas al núcleo familiar y no convivir con la madre. A medida que aumenta la severidad de la alteración de la conducta alimentaria, las adolescentes describen a sus familias con menor cohesión, mayor conflictividad y control; se evidencia también un aumento progresivo de síntomas psicopatológicos.<br/>Las adolescentes con TCANE son más corpulentas que el resto, el 77,3% de ellas y el 40% de las adolescentes a riesgo refieren elevada insatisfacción corporal. <br/>Hay una disminución progresiva en el consumo de alimentos, energía y macronutrientes en relación a la severidad de la alteración de la conducta alimentaria. Las adolescentes con riesgo de TCA y las adolescentes con TCANE consumen respectivamente alrededor de 500 y 900 Kcal. menos que las controles. <br/>La probabilidad de ingesta inadecuada es superior en las adolescentes a riesgo y las adolescentes con TCANE. El 67% y el 86% respectivamente realizan ingestas de calcio inferiores a las recomendaciones. Hay una mayor probabilidad de ingesta vitamínica inadecuada en adolescentes con TCANE que puede estar relacionado con las alteraciones mentales asociadas y podría ser un factor más que contribuyera a dar continuidad a los TCA.<br/>La restricción calórica es un factor de riesgo para cualquier nivel de severidad de alteración de la conducta alimentaria. El riesgo de síntomas de TCA se relaciona con las variables ansiedad y que algún familiar haga dieta, mientras que el riesgo de TCANE se relaciona con un IMC elevado, insatisfacción corporal y una situación de estrés (divorcio padres).<br>OBJETIVES:<br/><br/>- To estimate the prevalence of eating disorders (ED) at a different levels of severity (diagnosed as anorexia and nervous bulimia and eating disorders not otherwise specified (EDNOS) and symptoms of ED) in teenagers from the town of Tarragona and surrounding areas of Tarragona.<br/>- To describe and analysed the varying degrees of severity of the EDs.<br/>- To evaluate the influence of the different factors which contribute to the risk of contracting EDs.<br/><br/>METHODS AND MATERIAL:<br/><br/> A cross sectional study in two phases of teenagers of both sexes (12-16 years) who study (1st ,2nd ESO) in 26 both publics and privates schools in the Tarragona region. First phase (n=2967): To evaluate the prevalence of EDs symptoms using two screening test: Eating Attitudes Test (EAT) and Youth's Inventory. Second phase: only females studied (n=295). With the results of the test two groups were selected those which fell into the risk category (EAT>25 or Youth's (+) for ED) and non-risk category of ED (controls); evaluating: body satisfaction and autoperception, sociocultural influence, family climate, psychopathologic symptoms, physical activity, knowledge, preferences and dietary habits using written questionnaires. Individually evaluated was: food consumption using 24-h recall, body composition and the interview "Schedules for Clinical Assessment in Neuropsychiatry" (SCAN) in version DSM-IV, ICD-10 was administered to obtain the diagnoses.<br/><br/>RESULTS:<br/>13,3-17,1% of participants were considered at risk of suffering from an ED (18,9-22,5% females, 7,6-11,7% males) depending on the screening test used. The education level of parents is lower in parents of those female teenagers with ED symptoms. Stressful family backgrounds are also associated with a greater risk of ED symptoms. <br/>The estimate prevalence of ED in females is 5,2% (CI 95%: 5-5,4), nervous anorexia (0,53%) (CI 95%: 0,5-0,55), bulimia (0,66%) (CI 95%: 0,63-0-68) and EDNOS (4 %) (CI 95%: 3,83-4,17 ). Socioeconomic and educational stages of the parents were not associated with ED. EDNOS were more prevalent in teenagers with stressful family situations: divorce, living with a step-parent or unrelated people, and not living with the birth mother. As well as those factors affecting the severity of eating disorders, other factors include teenagers descriptions of the closeness of family, family conflicts and control; the evidence also notes the increase of psychopathologic symptoms. <br/>EDNOS are more frequently found among heavily-built teenage girls, 77,3% of those and the 40% of the teenagers at risk of ED reported high body dissatisfaction. <br/>There is a steady decline in the daily intake of food, energy and nutrients directly related to the severity of ED. The teenagers at risk of ED y the adolescents with EDNOS consume respectively around 500 to 900 Kcal. less than the controls. <br/>The probability of unsuitable intake is higher in those teenagers at risk of EDs and those with EDNOS. 67% and 86% respectively consume less than the recommended amount of calcium. There is a higher probability of unsuitable vitamin intake in adolescents with EDNOS that could be related to mental factors and this in itself could be another factor which contributes to the continuation of EDs. <br/>Caloric restraint is a risk factor that applies to all levels of ED. The risk of symptoms of EDs are directly related to factors such as anxiety, and to have any relative on diet; while the risk of EDNOS is related to a high body mass index (BMI), body dissatisfaction and stressful events (divorced parents).

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica<br>Enhanced biological phosphorus removal (EBPR) can be applied in wastewater treatment plants (WWTPs), as a sustainable and efficient way to remove phosphorus from wastewater and hence reduce its impact on eutrophication. This work characterises the performance, metabolism and identity of the microbial EBPR communities in full-scale WWTPs. The accurate quantification of the internal storage compounds, namely polyhydroxyalkanoate (PHA) and glycogen, is crucial to the characterisation of EBPR. The optimal glycogen and PHA quantification methods were sensitive to the heterogeneity of the sample, in terms of its microbial structure (floccular or granular) and, for PHA, in terms of the size and the number of substituents of the monomers forming the copolymer. Additionally, by characterising six full-scale EBPR WWTPs, in terms of their overall performance, microbial identity and metabolism, the composition of polyphosphate accumulating organisms (PAOs) was fairly similar in all plants. Also, a warmer climate was not sufficient to justify a higher presence of glycogen accumulating organisms (GAOs). Differing levels of denitrifying PAOs were obtained in different plants and the involvement of the tricarboxylic acid (TCA) cycle in the anaerobic metabolism of PAOs was observed. Furthermore, a metabolic model developed in this study, which incorporates the involvement of the anaerobic TCA cycle and a new description of the aerobic maintenance processes, was able to accurately describe the chemical cycling of soluble and intracellular compounds, while requiring a simple calibration procedure. A series of simulations demonstrated that lower acetate concentrations in the feed and higher aeration retention times would favour the TCA cycle metabolism over the glycolysis pathway, which would explain why the former has been more frequently encountered in WWTPs and the latter in lab-scale enriched cultures.<br>Fundação para a Ciência e Tecnologia - PhD grant (SFRH/BD/29477/2006)

La tesi realitza una prospectiva teòrica al voltant de la possibilitat de prevenir els trastorns del comportament alimentari (TCA) en nens i nenes d’Educació Primària –6 i 12 anys– a través de la televisió. Per un cantó, perquè les dades epidemiològiques ens alerten l’expansió dels TCA en edats que anteriorment eren respectades com els nens i nenes de Primària. Al mateix temps, són preocupants les pràctiques inadequades pel que fa a la conducta alimentària dels infants, que si bé sovint resten en mers trastorns subclínics, propicien que en un futur no gaire llunyà tinguin molta probabilitat de contraure algun TCA. I per altra, perquè, la televisió és un mitjà que malgrat sigui emprada com a bou expiatori de tots els mals de la nostra societat, té una gran potència educadora i un elevat consum social en els nens i nenes; així com en la societat en general. Per tant veiem que la complexitat que envolta els TCA així com la seva forta càrrega axiològica, ens suggereix que cal intervenir sobre els agents causants més educables. A tal efecte, a la present tesi doctoral s’efectua un repàs tant dels trets diagnòstics dels TCA, els principals models preventius, les diferents intervencions preventives en l’àmbit dels TCA i en altres problemàtiques de salut, que ens plantegen que és possible i necessari intervenir educativament a través de la televisió malgrat que no s’obviïn les dificultats que són inherents a la natura del mitjà televisiu. Per tant, es palesa la pertinència de contribuir a prevenir els TCA a través de la televisió. Ja que aquest esdevé una potent eina que educa tan als infants com a tot l’ecosistema que té capacitat d’exercir influència educativa en els nenes i nenes.<br>The thesis makes a theoretical searching about the possibility of prevention Eating Disorders (ED) on first education children –from 6 to 12 years old- using TV programs. On one hand, because epidemiological data warns expansion of ED in ages that once were respected as the children in that ages. At the same time, we have to worry the non-adequate alimentary practices referring to children alimentation, usually are non clinic problems but can be the beginning of a non far future ED serious problem. On the other hand, although TV is been used to justify many social problems, it has a great educational power and a high social consumption among children and general society. Thus we see that the complexity around ED as much as their high axiological dimension, make us think we should work on the most educable causal agents. That is why this doctoral thesis makes an overview about the diagnostic criteria of ED, main models of prevention, different preventive actions related to ED and any other health problems. We think that is possible to use television as an educational tool even we realize it has many lacks because of its own nature. That is why it is clear that television should, and must, be used to prevent ED. Because it’s a powerful tool which educates not only children but all their environment which can exercise a positive educational influence on them.

Orientador: Shiguenoli Miyamoto<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Filosofia e Ciencias Humanas<br>Made available in DSpace on 2018-08-07T10:06:38Z (GMT). No. of bitstreams: 1 Antiquera_DanieldeCampos_M.pdf: 1223976 bytes, checksum: b8117290cd5c1f3a54124d4c42ef46dd (MD5) Previous issue date: 2006<br>Resumo: Este trabalho analisa o Tratado de Cooperação Amazônica, assinado por Brasil, Bolívia, Colômbia, Equador, Guiana, Peru, Suriname e Venezuela em 1978, e suas transformações até se converter numa organização internacional (2002). O estudo busca relacionar essas mudanças no TCA com os diferentes momentos da política externa brasileira, visando em primeiro lugar a avaliar a importância da Amazônia para a política exterior do Brasil, e também a compreender o peso que a posição brasileira representa na evolução do Tratado. A partir do fim da década de 1980 há uma renovação no impulso dado ao TCA, que culmina com o protocolo de emenda, assinado em 1998, e a instalação de uma secretaria permanente, em Brasília, em 2002. Tal processo tem poucos estudos, embora indique uma renovação de expectativas sobre o Tratado. O trabalho foi feito por meio da análise de bibliografia sobre o TCA e a política externa, bem como sobre documentação oficial de ambas: discursos, atas de reuniões, declarações, estrutura jurídica. As informações foram complementadas com entrevistas realizadas com atores relevantes para o andamento do Tratado. A contribuição que este trabalho possa vir a dar é acrescentar outra perspectiva às leituras tradicionalmente liberais (no sentido de centrarem-se na cooperação e nos interesses comuns) que em geral são feitas sobre o objeto. Ao mesmo tempo, pretende-se chamar a atenção para a Amazônia e o norte da América do Sul como um espaço específico e importante das relações internacionais do Brasil<br>Abstract: This work analyzes The Amazon Cooperation Treaty, signed down by Brazil, Bolivia, Colombia, Equador, Guiana, Peru, Suriname e Venezuela in 1978, and its transformations until converting into international organization (2002). The study intends to establish a relation between those changes on ACT and different moments in brazilian foreign policy, looking for value amazonian role in this policy as well as for the brazilian role on the evolution of the Treaty. Since the end of the 80¿s, new strengths focused on ACT lead up to a renew¿s protocol, signed in 1998, and a permanent secretariat at Brasília, in 2002. There are few studies about these processes even than it indicates new expectations about the Treaty. The work was made analyzing the bibliography of ACT and of the foreign policy, as well as the official documentation of both of them. Also interviews have been made with important people involved in the Treaty. The eventual contribution of these research is offering a new perspective of the usually liberals (that focus on cooperation and common interests) points of view of the study¿s object. At the same time, there is the intention of getting attention for Amazon and for the north of South America as important areas of brazilian¿s international relations<br>Mestrado<br>Política Externa<br>Mestre em Relações Internacionais

An important cause of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To gain a better understanding of the disease process, the changes in biochemical pathways and metabolism of pulmonary arterial smooth muscle cells (PASMCs) were studied using a unique surgical ovine model of increased pulmonary blood flow. PASMCs isolated from 4-week-old lambs with increased PBF (shunt) showed lower oxygen consumption rates and lower extracellular acidification rates linked to glutamine metabolism when compared to controls. Shunt and control PASMCs both exhibited a switch into the reverse tricarboxylic acid (TCA) cycle, while only shunt cells showed a decrease of glucose being transformed into Acetyl CoA to enter the forward TCA cycle. Shunt PASMCs also demonstrated increased levels of yes-associated protein 1 (YAP1) expression in the nucleus. These results indicate changes in glutamine metabolism, glucose metabolism, and protein signaling cascades associated with increased mechanical forces in the setting of increased PBF, as seen in PAH in children with CHD.

Orientador: Cassio Odnei Garcia Munhoz<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba<br>Made available in DSpace on 2018-07-19T06:21:43Z (GMT). No. of bitstreams: 1 Beraldo_LuizFernando_M.pdf: 1150153 bytes, checksum: e36d9ebc0425ffdd1bfe51315122c5c3 (MD5) Previous issue date: 1993<br>Resumo: Neste estudo, procurou-se, através do método radioautográfico, observar os efeitos do EDT A a 4,13% e do TCA a 5% sobre as proteínas contidas nas secreções das células acinosas do pâncreas, nas células seromucosas da glândula submandibular e sobre a ultra-estrutura dessas glândulas de camundongos injetados com uma única dose de 3H-glicina e sacrificados uma hora após a injeção. As glândulas de cada animal foram divididas em pequenos fragmentos e estes, separados em 3 grupos: controles (não tratados), tratados pelo TCA e tratados com EDTA. As reações radioautográficas foram quantificadas mediante a contagem de grãos de prata reduzida sobre a secreção localizada na porção apical das células pancreáticas e sobre a secreção das células seromucosas da glândula submandibular. Os dados obtidos após as contagens dos grãos de prata mostraram, após a análise de variânciB, que não há diferenças significantes entre os três grupos. Conclui-se, assim, que tanto o TCA como o EDT A não provocam extrações de proteínas marcadas nas secreções. O TCA tem sobre o EDT A a vantagem de remover os sais minerais num intervalo de tempo mais curto, porém, a obtenção dos cortes semi-fmos no material tratado por ele é bem mais dificil. Ao microscópio eletrônico, o material tratado pelo EDT A apresenta o mesmo aspecto do controle. O TCA, ao contrário, provocou muitas alterações da ultra-estrutura de vários componentes celulares. Ao contrário do EDT A, seu uso como agente desmineralizador de tecidos duros em estudos ultra-estruturais não deve ser recomendado<br>Abstract: The aim of this study was to observe radioautographicaIly the effects of TCA and EDT A on the secretory proteins of the serous pancreatic cells, on the seromucous submandibular gland cells and on the ultrastructure of these glands of mice sacrificed lh afier the injection of a single dose of 3H-glicine by intracardiac fixation with 4% p-formaldehyde solution. The glands of each animal were divided in smaIl fragments (about lmm3) which were separated in three groups: control (non treated), treated with TCA and 1reated with EDT A. The radioautographic reactions were quantified by counting the reduced silver grains over the secretion of the serous pancreatic and seromucous submandibular gland cells. The counting data, afier statistical variance analysis, showed that both TCA and EDT A did not extract the labelled proteins of the acinar cells secretions of pancreas and submandibular glands. Although TCA removed the mineraIs faster than the EDT A it was more difficult to obtain ul1ra-thin and semi-thin sections in the gland fragments treated with it. Also the staining of the acinar cells with methilene blue was impaired. . The fragments treated by the EDT A did not present significant ul1rastructural differences when compared with controls. The TCA, on the other hand, caused some damage to the fine structure of various cellular components. Therefore, its use as a decalcitier of hard tiSsues on ultrastructural studies should not be recommended.<br>Mestrado<br>Histologia e Embriologia<br>Mestre em Biologia Buco-Dental

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-02-05T15:35:03Z No. of bitstreams: 1 Dissertação - Fabiane V. Soares.pdf: 2842986 bytes, checksum: f65acb16091242753d91f534b2d8d8f4 (MD5)<br>Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-02-05T15:35:37Z (GMT) No. of bitstreams: 1 Dissertação - Fabiane V. Soares.pdf: 2842986 bytes, checksum: f65acb16091242753d91f534b2d8d8f4 (MD5)<br>Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-02-05T15:36:24Z (GMT) No. of bitstreams: 1 Dissertação - Fabiane V. Soares.pdf: 2842986 bytes, checksum: f65acb16091242753d91f534b2d8d8f4 (MD5)<br>Made available in DSpace on 2016-02-05T15:36:24Z (GMT). No. of bitstreams: 1 Dissertação - Fabiane V. Soares.pdf: 2842986 bytes, checksum: f65acb16091242753d91f534b2d8d8f4 (MD5) Previous issue date: 2012-04-24<br>CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>The American Cutaneous Leishmaniasis (ACL) is a public health problem worldwide. It is widely distributed in Brazil and the Amazonas population is among the states most affected by the disease in the country. This study aimed to investigate the epidemiological and entomological leishmaniasis in the municipality of Tabatinga, Amazonas state. The city is situated on the left margin of the Solimões River, the border region to Colombia and Peru. We conducted a retrospective study (seven years), human cases of cutaneous leishmaniasis reported in the city, from data supplied by the Municipal Health Tabatinga and fauna survey of sand flies in August 2010 and 2011. Sandfly collections were carried out on the road from the National Institute of Colonization and Agrarian Reform - INCRA, Geodesy II, between km 4 and 8, using light traps (CDC) and aspiration in base tree with modified CDC. For statistical analysis we used the abundance index of species (ISA), standardized index of abundance of species (SISA), Index of Shannon-Wiener diversity (H ') and evenness index (J'). Have been reported 41 autochthonous cases of ACL in Tabatinga, where 61.7% were male, aged 30-39 years (24.4%) and farmers (21.9%). The predominant clinical form was cutaneous. We collected 5741 specimens representing 35 species of the genus Lutzomyia, both captured in the sylvatic and peridomiliciar environment. The most abundant species was L. umbratilis (SISA = 1.000). Of this total, 13 species are considered vectors or potential vectors of Leishmania and/or other trypanosomatids. Of the 44 positive samples, 10 were isolated / maintained in culture, eight from L. umbratilis and two L. dendrophyla. Isozyme analysis of these isolates showed similar profiles to Leishmania colombiensis and Endotrypanum sp., however, the profile of MDH locus showed greater similarity to the genus Endotrypanum. It made the report of the occurrence of two new species of sand flies in the Amazonas State (Lutzomyia howardi) and Brazil (Lutzomyia witoto). These are the first information on the phlebotomine fauna in the municipality of Tabatinga and expanding knowledge of the distribution, diversity and natural infection of trypanosomatids in the state of Amazonas<br>A Leishmaniose Tegumentar Americana (LTA) é um problema de Saúde Pública mundial. É amplamente distribuída no Brasil e a população amazonense encontra-se entre os estados mais acometidos pela doença no país. Este estudo teve como objetivo averiguar os aspectos epidemiológicos e entomológicos da LTA no município de Tabatinga, Estado do Amazonas. O município está situado à margem esquerda do rio Solimões, região fronteiriça à Colômbia e ao Peru. Foi realizado um estudo retrospectivo (últimos sete anos) dos casos humanos de leishmaniose cutânea notificados no município, a partir dos dados fornecidos pela Secretaria Municipal de Saúde de Tabatinga e o levantamento da fauna de flebotomíneos em agosto de 2010 e 2011. As coletas de flebotomíneos foram realizadas na Estrada do Instituto Nacional de Colonização e Reforma Agrária - INCRA, Geodésica II, entre os km 4 e 8, utilizando-se armadilhas luminosas (CDC) e aspiração em base de árvore com CDC modificada. Para análise estatística foram utilizados o Índíce de Abundância de Espécies (IAE), Índíce Padronizado de Abundância de Espécies (IPAE), Índice de Diversidade Shannon-Wiener (H’) e Índice de eqüitabilidade (J’). Foram notificados 41 casos autóctones de LTA em Tabatinga, onde 61,7% eram do sexo masculino, com faixa etária de 30 a 39 anos (24,4%) e agricultores (21,9%). A forma clínica predominantemente foi cutânea. Foram coletados 5.741 espécimens, correspondente a 35 espécies do gênero Lutzomyia, capturados tanto em ambiente silvestre quanto peridomiliciar. A espécie mais abundante foi L. umbratilis (IPAE= 1.000). Deste total, 13 espécies são consideradas transmissoras ou vetores potenciais de Leishmania e/ou outros tripanosomatídeos. Das 44 amostras positivas, 10 foram isoladas/mantidas em cultivo, sendo oito de L. umbratilis e duas de L. dendrophyla. Análise isoenzimática desses isolados, demonstrou perfis similares a Leishmania colombiensis e Endotrypanum sp., no entanto, o perfil do locus MDH demonstrou maior similaridade para o gênero Endotrypanum. Foi feito o relato da ocorrência de duas novas espécies de flebótomos no Amazonas (Lutzomyia howardi) e no Brasil (Lutzomyia witoto). Os resultados deste trabalho referem-se à primeira informação sobre a fauna de flebotomíneos no município de Tabatinga, ampliando o conhecimento da distribuição, diversidade e infecção natural por tripanosomatídeos no Estado do Amazonas.